The chemiluminescence based Ziplex® automated workstation focus array reproduces ovarian cancer Affymetrix GeneChip® expression profiles

  • Michael CJ Quinn1,

    Affiliated with

    • Daniel J Wilson2,

      Affiliated with

      • Fiona Young2,

        Affiliated with

        • Adam A Dempsey2,

          Affiliated with

          • Suzanna L Arcand3,

            Affiliated with

            • Ashley H Birch1,

              Affiliated with

              • Paulina M Wojnarowicz1,

                Affiliated with

                • Diane Provencher4, 5, 6,

                  Affiliated with

                  • Anne-Marie Mes-Masson4, 6,

                    Affiliated with

                    • David Englert2 and

                      Affiliated with

                      • Patricia N Tonin1, 3, 7Email author

                        Affiliated with

                        Journal of Translational Medicine20097:55

                        DOI: 10.1186/1479-5876-7-55

                        Received: 07 April 2009

                        Accepted: 06 July 2009

                        Published: 06 July 2009

                        Abstract

                        Background

                        As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex® technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip® analyses.

                        Methods

                        The new chemiluminescence-based Ziplex® gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip® profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing.

                        Results

                        Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement.

                        Conclusion

                        Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

                        Background

                        During the last decade, the advent of high-throughput techniques such as DNA microarrays, has allowed investigators to interrogate the expression level of thousands of genes concurrently. Due to the heterogeneous nature of many cancers in terms of both their genetic and molecular origins and their response to treatment, individualizing patient treatment based on the expression levels of signature genes may impact favorably on patient management [1, 2]. In ovarian cancer, discrete gene signatures have been determined from microarray analysis of ovarian cancer versus normal ovarian tissue [36], correlating gene expression profiles to survival or prognosis [7, 8], studies of chemotherapy resistance [9, 10], and functional studies such as chromosome transfer experiments [11, 12]. Recent studies have focused on a biomarker approach [13], with specific prognostic markers being discovered by relating gene expression profiles to clinical variables [1416]. In addition, there is a trend towards offering patient-tailored therapy, where expression profiles are related to key clinical features such as TP53 or HER2 status, surgical outcome and chemotherapy resistance [1, 17].

                        A major challenge in translating promising mRNA-based expression biomarkers has been the reproducibility of results when adapting gene expression assays to alternative platforms that are specifically developed for clinical laboratories. Xceed Molecular has recently developed a multiplex gene expression assay technology termed the Ziplex® Automated Workstation, designed to facilitate the expression analysis of a discrete number of genes (up to 120) specifically intended for clinical translational laboratories. The Ziplex array is essentially a three-dimensional array comprised of a microporous silicon matrix containing oligonucleotides probes mounted on a plastic tube. The probes are designed to overlap the target sequences of the probes used in large-scale gene expression array platforms from which the expression signature of interest was initially detected, such as the 3' UTR target sequences of the Affymetrix GeneChip®. However unlike most large-scale expression platforms, gene expression detection is by chemiluminescence. Recently, the Ziplex technology was compared to five other commercially available and well established gene expression profiling systems following the methods introduced by the MicroArray Quality Control (MAQC) consortium [1820] and reported in a white paper by Xceed Molecular [21]. The original MAQC study (MAQC Consortium, 2006) was undertaken because of concerns about the reproducibility and cross-platform concordance between gene expression profiling platforms, such as microarrays and alternative quantitative platforms. By assessing the expression levels of the MAQC panel of 53 genes on universal RNA samples, it was determined that the reproducibility, repeatability and sensitivity of the Ziplex system were at least equivalent to that of other MAQC platforms [21].

                        There is a need to implement reliable gene expression technologies that are readily adaptable to clinical laboratories in order to screen individual or multiple gene expression profiles ("signature") identified by large-scale gene expression assays of cancer samples. Our ovarian cancer research group (as well as other independent groups) has identified specific gene expression profiles from mining Affymetrix GeneChip expression data illustrating the utility of this approach at identifying gene signature patterns associated with specific parameters of the disease [14, 22]. Ovarian cancer specimens are typically large and exhibit less tumor heterogeneity and thus may be amenable to gene expression profiling in a reproducible way. However, until recently the gene expression technologies available that could easily be adapted to a clinical setting have been limited primarily by the expertise required to operate them. The recently developed Ziplex Automated Workstation offers a opportunity to develop RNA expression-based biomarkers that could readily be adapted to clinical settings as the 'all-in-one' technology appears to be relatively easy to use. However, this system has not been applied to ovarian cancer disease nor has its use been reported in human systems. In the present study we have evaluated the reproducibility of the Ziplex system using 93 genes, selected based on their expression profile as initially assessed by Affymetrix GeneChip microarray analyses from a number of ovarian cancer research studies from our group [6, 14, 2226]. These include genes which are highly differentially expressed between ovarian tumor samples and normal ovary samples that were identified using both newer and older generation GeneChips [6, 22, 25, 26]. In addition, to address the question of sensitivity, genes known to have a wide range of expression values were tested some of which show comparable values of expression between representative normal and ovarian tumor tissue samples but represent a broad range of expression values [25, 26]. Other genes known to be relevant to ovarian cancer including tumor suppressor genes and oncogenes were included in the analysis. Selected highly differentially expressed genes from an independent microarray analysis of ovarian tumors compared to short term cultures of normal epithelial cells was also included [3]. In many cases, the level of gene expression identified by Affymetrix GeneChip analysis was independently validated by semi-quantitative RT-PCR, real-time RT-PCR, or Northern Blot analysis [6, 14, 22, 2426]. Expression assays were performed using RNA from serous ovarian tumors, short term cultures of normal ovarian surface epithelial cells, and four well characterized ovarian cancer cell lines which were selected based on their known expression profiles using Affymetrix microarray analyses. Comparisons were made between the Ziplex system and expression profiles generated using the U133A Affymetrix GeneChip platform. An important aspect of this study was that gene expression profiling of Ziplex system was performed in a blinded fashion where the sample content was not known to the immediate users. It is envisaged that both the nature of the candidates chosen and their range of gene expression will permit for a direct comment on the sensitivity, reproducibility and overall utility of the Ziplex array as a platform for gene expression array analysis for translational research.

                        Methods

                        Source of RNA

                        Total RNA was extracted with TRIzol reagent (Gibco/BRL, Life Technologies Inc., Grand Island, NY) from primary cultures of normal ovarian surface epithelial (NOSE) cells, frozen malignant serous ovarian tumor (TOV) samples and epithelial ovarian cancer (EOC) cell lines as described previously [27]. Additional File 1 provides a description of samples used in the expression analyses.

                        The NOSE and TOV samples were attained from the study participants at the Centre de recherche du Centre hospitalier de l'Université de Montréal – Hôpital Hotel-Dieu and Institut du cancer de Montréal with signed informed consent as part of the tissue and clinical banking activities of the Banque de tissus et de données of the Réseau de recherche sur le cancer of the Fonds de la Recherche en Santé du Québec (FRSQ). The study was granted ethical approval from the Research Ethics Boards of the participating research institutes.

                        Ziplex array and probe design

                        The 93 genes used for assessing the reproducibility of the Ziplex array are shown in Table 1. The criteria for gene selection were: genes exhibiting statistically significant differential expression between NOSE and TOV samples as assessed by Affymetrix U133A microarray analysis; genes exhibiting a range of expression values (nominally low, medium or high) based on Affymetrix U133A microarray analysis, in order to assess sensitivity; genes exhibiting differential expression profiles based on older generation Affymetrix GeneChips (Hs 6000 [6] and Hu 6800 [23]); and genes known or suspected to play a role in ovarian cancer (Table 1). Initial selection criteria for genes in their original study included individual two-way comparisons [25, 26], fold-differences [6, 23], and fold change analysis using SAM (Significance Analysis of Microarrays) [3] between TOV and NOSE groups. Some genes were selected based on their low, mid or high range of expression values that did not necessarily exhibit statistically significant differences between TOV and NOSE groups.
                        Table 1

                        Selection Criteria of Genes Assayed by Ziplex Technology

                        Selection Criteria Categories

                        Affymetrix U133A Probe Set

                        GeneID*

                        Gene Name

                        Reference

                        A: Differentially expressed genes based on Affymetrix U133A analysis

                        208782_at

                        11167

                        FSTL1

                        25

                         

                        213069_at

                        57493

                        HEG1

                        25

                         

                        218729_at

                        56925

                        LXN

                        25

                         

                        202620_s_at

                        5352

                        PLOD2

                        25

                         

                        217811_at

                        51714

                        SELT

                        25

                         

                        213338_at

                        25907

                        TMEM158

                        25

                         

                        203282_at

                        2632

                        GBE1

                        25

                         

                        204846_at

                        1356

                        CP

                        25

                         

                        221884_at

                        2122

                        EVI1

                        25

                         

                        202310_s_at

                        1277

                        COL1A1

                        26

                         

                        201508_at

                        3487

                        IGFBP4

                        26

                         

                        200654_at

                        5034

                        P4HB

                        26

                         

                        212372_at

                        4628

                        MYH10

                        26

                         

                        216598_s_at

                        6347

                        CCL2

                        26

                         

                        208626_s_at

                        10493

                        VAT1

                        26

                         

                        41220_at

                        10801

                        SEPT9

                        26

                         

                        208789_at

                        284119

                        PTRF

                        26

                         

                        206295_at

                        3606

                        IL18

                        22

                         

                        202859_x_at

                        3576

                        IL8

                        22

                         

                        209969_s_at

                        6772

                        STAT1

                        22

                         

                        209846_s_at

                        11118

                        BTN3A2

                        22

                         

                        220327_at

                        389136

                        VGLL3

                        11

                         

                        203180_at

                        220

                        ALDH1A3

                        26

                         

                        204338_s_at

                        5999

                        RGS4

                        26

                         

                        204879_at

                        10630

                        PDPN

                        26

                         

                        207510_at

                        623

                        BDKRB1

                        26

                         

                        208131_s_at

                        5740

                        PTGIS

                        26

                         

                        211430_s_at

                        3500

                        IGHG1

                        26

                         

                        216834_at

                        5996

                        RGS1

                        26

                         

                        266_s_at

                        100133941

                        CD24

                        26

                         

                        213994_s_at

                        10418

                        SPON1

                        26

                         

                        221671_x_at

                        3514

                        IGKC

                        26

                        B: Genes exhibiting a range of expression values based on Affymetrix U133A analysis

                        218304_s_at

                        114885

                        OSBPL11

                        25

                         

                        219295_s_at

                        26577

                        PCOLCE2

                        25

                         

                        205329_s_at

                        8723

                        SNX4

                        25

                         

                        219036_at

                        80321

                        CEP70

                        25

                         

                        218926_at

                        55892

                        MYNN

                        25

                         

                        208836_at

                        483

                        ATP1B3

                        25

                         

                        204992_s_at

                        5217

                        PFN2

                        25

                         

                        214143_x_at

                        6152

                        RPL24

                        25

                         

                        208691_at

                        7037

                        TFRC

                        25

                         

                        203002_at

                        51421

                        AMOTL2

                        25

                         

                        221492_s_at

                        64422

                        ATG3

                        25

                         

                        218286_s_at

                        9616

                        RNF7

                        25

                         

                        212058_at

                        23350

                        SR140

                        25

                         

                        201519_at

                        9868

                        TOMM70A

                        25

                         

                        209933_s_at

                        11314

                        CD300A

                        26

                         

                        219184_x_at

                        29928

                        TIMM22

                        26

                         

                        204683_at

                        3384

                        ICAM2

                        26

                         

                        212529_at

                        124801

                        LSM12

                        26

                         

                        211899_s_at

                        9618

                        TRAF4

                        26

                         

                        218014_at

                        79902

                        NUP85

                        26

                         

                        200816_s_at

                        5048

                        PAFAH1B1

                        26

                         

                        202395_at

                        4905

                        NSF

                        26

                         

                        201388_at

                        5709

                        PSMD3

                        26

                         

                        220975_s_at

                        114897

                        C1QTNF1

                        26

                         

                        210561_s_at

                        26118

                        WSB1

                        26

                         

                        202856_s_at

                        9123

                        SLC16A3

                        26

                         

                        212279_at

                        27346

                        TMEM97

                        26

                         

                        37408_at

                        9902

                        MRC2

                        26

                         

                        201140_s_at

                        5878

                        RAB5C

                        26

                         

                        214218_s_at

                        7503

                        XIST

                        24

                         

                        200600_at

                        4478

                        MSN

                        24

                         

                        201136_at

                        5355

                        PLP2

                        24

                        C: Genes exhibiting differential expression profiles based on older generation Affymetrix GeneChips (Hs 6000 (6), Hu 6800 (22))

                        202431_s_at

                        4609

                        MYC

                        6

                         

                        203752_s_at

                        3727

                        JUND

                        6

                         

                        205009_at

                        7031

                        TFF1

                        6

                         

                        205067_at

                        3553

                        IL1B

                        6

                         

                        200807_s_at

                        3329

                        HSPD1

                        6

                         

                        203139_at

                        1612

                        DAPK1

                        6

                         

                        200886_s_at

                        5223

                        PGAM1

                        6

                         

                        203083_at

                        7058

                        THBS2

                        6

                         

                        202284_s_at

                        1026

                        CDKN1A

                        6

                         

                        212667_at

                        6678

                        SPARC

                        6

                         

                        202627_s_at

                        5054

                        SERPINE1

                        6

                         

                        203382_s_at

                        348

                        APOE

                        6

                         

                        211300_s_at

                        7157

                        TP53

                        6

                         

                        200953_s_at

                        894

                        CCND2

                        6

                         

                        201700_at

                        896

                        CCND3

                        6

                         

                        205881_at

                        7625

                        ZNF74

                        23

                         

                        207081_s_at

                        5297

                        PI4KA

                        23

                         

                        205576_at

                        3053

                        SERPIND1

                        23

                         

                        203412_at

                        8216

                        LZTR1

                        23

                         

                        206184_at

                        1399

                        CRKL

                        23

                        D: Known oncogenes and tumour U133A analysis suppressor genes relevant to ovarian cancer biology

                        203132_at

                        5925

                        RB1

                         
                         

                        204531_s_at

                        672

                        BRCA1

                         
                         

                        214727_at

                        675

                        BRCA2

                         
                         

                        202520_s_at

                        4292

                        MLH1

                         
                         

                        216836_s_at

                        2064

                        ERBB2

                         
                         

                        204009_s_at

                        3845

                        KRAS

                         
                         

                        206044_s_at

                        673

                        BRAF

                         
                         

                        209421_at

                        4436

                        MSH2

                         
                         

                        211450_s_at

                        2956

                        MSH6

                         

                        *GeneID (gene identification number) is based on the nomenclature used in the Entrez Gene database available through the National Center for Biotechnology Information (NCBI)

                        http://​www.​ncbi.​nlm.​nih.​gov.

                        The Ziplex array or TipChip is a three-dimensional array comprised of a microporous silicon matrix containing oligonucleotide probes that is mounted on a plastic tube. Each probe was spotted in triplicate. In order to replicate gene expression assays derived from the Affymetrix GeneChip analysis, probe set design was based on the Affymetrix U133A probe set target sequences for the selected gene (refer to Table 1). Gene names were assigned using UniGene ID Build 215 (17 August 2008). To improve accuracy of probe design, and to account for variation of probe hybridization, up to three probes were designed for each gene. From this exercise, a single probe was chosen to provide the most reliable and consistent quantification of gene expression. Gene accession numbers corresponding to the Affymetrix probe set sequences for each gene were verified by BLAST alignment searches of the NCBI Transcript Reference Sequences (RefSeq) database http://​www.​ncbi.​nlm.​nih.​gov/​projects/​RefSeq/​. Array Designer (Premier Biosoft, Palo Alto, CA) was used to generate three probes from each verified RefSeq transcript that were between 35 to 50 bases in length (median 46 base pairs), exhibited a melting temperature of approximately 70°C, represent a maximum distance of 1,500 base pairs from the from 3' end of the transcript, and exhibited minimal homology to non-target RefSeq sequences. Using this approach it was possible to design three probes for 92 of the 93 selected genes: APOE was represented by only two probes. For the 93 genes analyzed, the median distance from the 3' end was 263 bases, whereas less than 12% of the probes were more than 600 bases from the 3' end. Ten probes were also designed for genes that were not expected to vary significantly between TOV and NOSE samples based on approximately equal expression in the two sample types and relatively low coefficients of variation (18 to 20%) as assessed by Affymetrix U133A microarray analysis of the samples; such probes were potential normalization controls. Based on standard quality control measures of the manufacturer, three probes representing ACTB, GAPDH, and UBC and a set of standard control probes, including a set of 5' end biased probes for RPL4, POLR2A, ACTB, GAPDH and ACADVL were printed on each array for data normalization and quality assessment. The probes were printed on two separate TipChip arrays.

                        Hybridization and raw data collection

                        Total RNA from NOSE and TOV samples and the four EOC cell lines were prepared as described above and provided to Xceed Molecular for hybridization and data collection in a blinded manner. RNA quality (RNA integrity number (RIN)) using the Agilent 2100 Bioanalyzer Nano, total RNA assay was assessed for each sample (Additional File 1). For each sample, approximately 500 ng of RNA was amplified and labeled with the Illumina® TotalPrep™ RNA Amplification Kit (Ambion, Applied Biosystems Canada, Streetsville, ON, CANADA). Although sample MG0026 (TOV-1150G) had a low RIN number, it was carried through the study. Sample MG0001 (TOV-21G) had no detectable RIN number and MG0013 (NOV-1181) failed to produce amplified RNA. Neither of these samples were carried through the study. Five μg of the resulting biotin-labeled amplified RNA was hybridized on each TipChip. The target molecules were biotin labeled, and an HRP-streptavidin complex was used for imaging of bound targets by chemiluminescence. Hybridization, washing, chemiluminescent imaging and data collection were automatically performed by the Ziplex Workstation (Xceed Molecular, Toronto, ON, Canada).

                        Data normalization

                        The mean ratio of the intensities of the replicate probes that were printed on both of the ovarian cancer arrays were used to scale the data between the two TipChip arrays hybridized with each sample. The mean scaling factor for the 27 samples was 1.03 with a maximum of 1.23. The coefficients of variation (CV) across 27 samples and the expression differences between NOSE and TOV samples was calculated from the raw data for each of the 10 genes included on the arrays as potential normalization genes (Additional File 2). The geometric means of the signals for probes for PARK7, PI4KB, TBCB, and UBC with small CVs (mean of 25%) and insignificant differences between NOSE and TOV (p > 0.48) were used to normalize the data (refer to Additional File 2 for all normalization gene results). The data were analyzed with and without normalization.

                        Selection of optimal probe design

                        The hybridization intensities of the replicate probes designed for each gene for the 27 samples were compared to choose a single probe per gene with optimal performance. This assessment was based on signal intensity (well above the noise level and within the dynamic range of the system), minimum distance from the 3' end of the target sequence and correlation between different probe designs. Minimum distance from the 3' end is a consideration since the RNA sample preparation process is somewhat biased to the 3' end of the transcripts. The signals for probes for the same target should vary proportionally between different samples if both probes bind to and only to the nominal target. Good correlation between different Ziplex probe designs for genes in the RefSeq database, as well as good correlation with the Affymetrix data and discrimination between sample types, infers that probes bind to the intended target sequences. Data from the chosen probe was used for all subsequent analysis. Correlations of signal intensities for pairs of probes for the same genes are presented in Additional File 3.

                        Comparative analysis of Ziplex and Affymetrix data

                        Correlations between Ziplex and Affymetrix array datasets were calculated. The Affymetrix U133A data was previously derived from RNA expression analysis of the NOSE and TOV samples and EOC cell lines. Hybridization and scanning was performed at the McGill University and Genome Quebec Innovation Centre http://​www.​genomequebecplat​forms.​com. MAS5.0 software (Affymetrix® Microarray Suite) was used to quantify gene expression levels. Data was normalized by multiplying the raw value for an individual probe set (n = 22,216) by 100 and dividing by the mean of the raw expression values for the given sample data set, as described previously [23, 28]. Affymetrix and Ziplex data were matched by gene, and correlations (p < 0.01, using values only of greater than 4) and a graphical representation was determined using Mathematica (Version 6.03) software (Wolfram Research, Inc., Champaign, IL, USA). Mean signal intensity values were log2 transformed and compared between NOSE and TOV data using a Welch Rank Sum Test, for both Affymetrix microarray and Ziplex array data. A p-value of less than 0.001 was used as the significance level.

                        Composition of mean-difference plots followed the method of Bland and Altman [29]. Briefly, the mean of the log2 fold change and the difference between the log2 fold change for the platforms under comparison were calculated and plotted. The 95% limits of agreement were calculated as follows: log2 fold change difference ± 1.96 × standard deviation of the log2 fold change difference.

                        Quality control of Ziplex array data

                        The percent CVs were greater for probes with signals below 30. The overall median of the median probe percent CV was 4.7%. The median of the median percent CVs was 4.4% for probes with median intensities greater than 30, and 8.0% for probes with median CVs less than or equal to 30. The signal to noise (SNR) values is the average of the ratios for the net signals of the replicate spots to the standard deviation of the pixel values used to evaluate background levels (an image noise estimate). Average SNR ranged from -0.3 to 32.8. The signal intensities and ratios of intensity signals derived from 3' and 5' probes are shown in Additional File 4. Sample MG0001, which included many high 3'/5' ratios, was not included for subsequent analysis. The 3'/5' signal intensity ratios correlated with the RIN numbers and 28 S/18 S ratios (Additional File 5), indicating that, as expected, amplified RNA fragment lengths vary according to the integrity of the total RNA sample.

                        Results

                        Correlation of Affymetrix U133A and Ziplex array expression profiles

                        Normalized Affymetrix U133A and Ziplex gene expression data were matched by gene. For each gene expression platform, values less than 4 were considered to contribute to censoring bias and were not included in the correlation analysis. Correlations (log10 transformed) for paired gene expression data ranged from 0.0277 to 0.998, with an average correlation of 0.811 between Affymetrix and Ziplex gene expression data (Additional File 6). For a detailed summary of the correlation analysis, see also Additional File 7. The expression profiles of 82 of the 93 (88.2%) genes were significantly positively correlated (p < 0.01) in a comparison of the two platforms. As shown with the selected examples, genes exhibiting under-expression, such as ALDH1A3 and CCL2, or over-expression, such as APOE and EVI1, in the TOV samples relative to the NOSE samples by Affymetrix U133A microarray analysis also exhibited similar patterns of expression by Ziplex array (Figure 1). In contrast, TRAF4 expression was not correlated between the platforms (R2 = 0.0003). However, both platforms yielded low expression values for this gene. Although gene expression at very low levels may be difficult to assay and can be affected by technical variability, a good correspondence between platforms can be achieved with specific probes, as shown in the comparison of the BRCA1 expression profiles (R2 = 0.870) (Figure 1).
                        http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-7-55/MediaObjects/12967_2009_Article_366_Fig1_HTML.jpg
                        Figure 1

                        Correlation plots of selected genes underexpressed in TOV (A, B), over-expressed in TOV (C, D) and showing low expression (E, F) across samples. Xceed Ziplex (XZP) expression data is plotted on the x axis and Affymetrix (AFX) microarray data on the y axis. The EOC cell lines are indicated in green (n = 3), TOV samples in red (n = 12) and NOSE samples in blue (n = 11). Correlation coefficients are shown at the bottom right.

                        Comparative analysis of fold changes of Affymetrix U133A and Ziplex array expression profiles

                        The fold change differences in gene expression were compared between the two platforms. There was a strong correspondence of gene expression patterns across the platforms when compared for each gene (Table 2). In terms of overall concordance of statistical significance between NOSE and TOV samples, there were consistent results for 75 of 93 genes by Affymetrix and Ziplex analysis (p < 0.001) by Welch rank sum test, in each platform. The fold change differences were concordant for 87 of 93 (94%) genes where there was agreement between the platforms regarding statistical significance for 71 (76%) of the 87 genes. The fold change differences were discordant for 6 genes, but the differences were statistically insignificant on both platforms for four of these genes. For example for the gene SERPIND1, there is no concordance in terms of fold change between the two platforms, but these fold change differences are not significant for either platform (p > 0.001). These results exemplifies that caution should be used when relying on fold change results alone. Notably, for two of the discordantly expressed genes (MSH6 and TFF1), the fold change differences were statistically significant (p < 0.001) only on the Ziplex platform but not for the Affymetrix platform.
                        Table 2

                        Comparison of mean signal intensity (SI) values for the 93 gene probes between NOSE and TOV samples

                          

                        Affymetrix U133A Array

                        Ziplex Automated Workstation

                        Platform Comparison

                        Selection Criteria1

                        Gene Probe

                        NOSE mean SI (n = 11)

                        TOV mean SI (n = 12)

                        ratio (N/T)2

                        ratio (T/N)2

                        p-value3

                        NOSE mean SI (n = 11)

                        TOV mean SI (n = 12)

                        ratio (N/T)2

                        ratio (T/N)2

                        p-value3

                        significance based on p-value3

                        concordance based on ratio fold-change direction

                        A

                        RGS4

                        291

                        2

                        181.2

                        0.01

                        <0.0001

                        863

                        41

                        21.1

                        0.05

                        <0.0001

                        agree

                        concordance

                        C

                        SERPINE1

                        1912

                        12

                        162.4

                        0.01

                        <0.0001

                        1426

                        17

                        82.2

                        0.01

                        <0.0001

                        agree

                        concordance

                        A

                        PDPN

                        57

                        2

                        23.9

                        0.04

                        0.0008

                        100

                        35

                        2.9

                        0.35

                        0.0023

                        disagree

                        concordance

                        A

                        ALDH1A3

                        661

                        29

                        22.6

                        0.04

                        0.0020

                        1887

                        76

                        24.8

                        0.04

                        0.0051

                        agree

                        concordance

                        A

                        IL8

                        1353

                        69

                        19.7

                        0.05

                        0.0151

                        4465

                        231

                        19.3

                        0.05

                        0.0015

                        agree

                        concordance

                        A

                        PTGIS

                        1470

                        80

                        18.4

                        0.05

                        <0.0001

                        3474

                        184

                        18.9

                        0.05

                        <0.0001

                        agree

                        concordance

                        A

                        HEG1

                        923

                        66

                        14.1

                        0.07

                        <0.0001

                        3184

                        252

                        12.6

                        0.08

                        <0.0001

                        agree

                        concordance

                        A

                        TMEM158

                        461

                        33

                        13.9

                        0.07

                        <0.0001

                        869

                        46

                        18.8

                        0.05

                        <0.0001

                        agree

                        concordance

                        C

                        CDKN1A

                        598

                        53

                        11.4

                        0.09

                        <0.0001

                        385

                        63

                        6.1

                        0.16

                        <0.0001

                        agree

                        concordance

                        A

                        CCL2

                        570

                        54

                        10.6

                        0.09

                        0.0010

                        1923

                        207

                        9.3

                        0.11

                        0.0001

                        agree

                        concordance

                        A

                        LXN

                        731

                        73

                        10.1

                        0.10

                        <0.0001

                        926

                        124

                        7.5

                        0.13

                        0.0002

                        agree

                        concordance

                        C

                        SPARC

                        1037

                        108

                        9.6

                        0.10

                        <0.0001

                        2841

                        341

                        8.3

                        0.12

                        <0.0001

                        agree

                        concordance

                        C

                        IL1B

                        666

                        70

                        9.6

                        0.10

                        0.0247

                        1559

                        46

                        34.0

                        0.03

                        0.0035

                        agree

                        concordance

                        A

                        BDKRB1

                        152

                        18

                        8.7

                        0.11

                        0.0004

                        464

                        22

                        21.0

                        0.05

                        <0.0001

                        agree

                        concordance

                        B

                        SLC16A3

                        425

                        63

                        6.8

                        0.15

                        <0.0001

                        197

                        37

                        5.3

                        0.19

                        <0.0001

                        agree

                        concordance

                        A

                        FSTL1

                        1837

                        277

                        6.6

                        0.15

                        <0.0001

                        5293

                        732

                        7.2

                        0.14

                        <0.0001

                        agree

                        concordance

                        C

                        THBS2

                        846

                        135

                        6.3

                        0.16

                        <0.0001

                        668

                        105

                        6.4

                        0.16

                        0.0009

                        agree

                        concordance

                        A

                        IGFBP4

                        1484

                        238

                        6.2

                        0.16

                        <0.0001

                        692

                        122

                        5.7

                        0.18

                        0.0001

                        agree

                        concordance

                        A

                        PTRF

                        976

                        168

                        5.8

                        0.17

                        <0.0001

                        217

                        77

                        2.8

                        0.35

                        <0.0001

                        agree

                        concordance

                        A

                        GBE1

                        775

                        136

                        5.7

                        0.18

                        <0.0001

                        988

                        173

                        5.7

                        0.17

                        <0.0001

                        agree

                        concordance

                        A

                        PLOD2

                        654

                        123

                        5.3

                        0.19

                        <0.0001

                        926

                        132

                        7.0

                        0.14

                        <0.0001

                        agree

                        concordance

                        A

                        VAT1

                        874

                        175

                        5.0

                        0.20

                        <0.0001

                        255

                        78

                        3.3

                        0.31

                        <0.0001

                        agree

                        concordance

                        A

                        COL1A1

                        2940

                        614

                        4.8

                        0.21

                        0.0001

                        1502

                        289

                        5.2

                        0.19

                        0.0003

                        agree

                        concordance

                        C

                        CCND2

                        324

                        70

                        4.7

                        0.21

                        0.0127

                        481

                        117

                        4.1

                        0.24

                        0.0337

                        agree

                        concordance

                        A

                        SELT

                        558

                        148

                        3.8

                        0.27

                        0.0010

                        166

                        137

                        1.2

                        0.8

                        >0.05

                        disagree

                        concordance

                        B

                        C1QTNF1

                        169

                        48

                        3.6

                        0.28

                        <0.0001

                        30

                        3

                        11.7

                        0.09

                        <0.0001

                        agree

                        concordance

                        A

                        VGLL3

                        35

                        10

                        3.5

                        0.29

                        <0.0001

                        75

                        12

                        6.1

                        0.16

                        0.0015

                        disagree

                        concordance

                        C

                        PGAM1

                        1482

                        473

                        3.1

                        0.32

                        <0.0001

                        1603

                        504

                        3.2

                        0.31

                        <0.0001

                        agree

                        concordance

                        C

                        TP53

                        55

                        18

                        3.0

                        0.33

                        0.0178

                        197

                        226

                        0.9

                        1.1

                        >0.05

                        agree

                        discordance

                        B

                        MSN

                        746

                        250

                        3.0

                        0.33

                        <0.0001

                        818

                        354

                        2.3

                        0.43

                        <0.0001

                        agree

                        concordance

                        B

                        PSMD3

                        196

                        66

                        3.0

                        0.34

                        <0.0001

                        735

                        384

                        1.9

                        0.5

                        <0.0001

                        agree

                        concordance

                        B

                        WSB1

                        300

                        103

                        2.9

                        0.34

                        0.0003

                        313

                        155

                        2.0

                        0.50

                        0.0006

                        agree

                        concordance

                        B

                        MRC2

                        313

                        109

                        2.9

                        0.35

                        <0.0001

                        528

                        138

                        3.8

                        0.26

                        <0.0001

                        agree

                        concordance

                        A

                        MYH10

                        1113

                        420

                        2.6

                        0.38

                        0.0006

                        1096

                        464

                        2.4

                        0.42

                        0.0106

                        disagree

                        concordance

                        B

                        NSF

                        180

                        72

                        2.5

                        0.40

                        <0.0001

                        304

                        170

                        1.8

                        0.6

                        0.0023

                        disagree

                        concordance

                        A

                        P4HB

                        2276

                        917

                        2.5

                        0.40

                        <0.0001

                        4567

                        1553

                        2.9

                        0.34

                        <0.0001

                        agree

                        concordance

                        C

                        SERPIND1

                        7

                        3

                        2.2

                        0.45

                        >0.05

                        79

                        117

                        0.7

                        1.5

                        0.0363

                        agree

                        discordance

                        B

                        RAB5C

                        309

                        142

                        2.2

                        0.46

                        0.0106

                        132

                        61

                        2.2

                        0.46

                        <0.0001

                        disagree

                        concordance

                        B

                        PFN2

                        800

                        392

                        2.0

                        0.49

                        <0.0001

                        699

                        444

                        1.6

                        0.6

                        0.0005

                        agree

                        concordance

                        B

                        TRAF4

                        47

                        23

                        2.0

                        0.50

                        0.0363

                        30

                        27

                        1.1

                        0.9

                        >0.05

                        agree

                        concordance

                        B

                        LSM12

                        59

                        31

                        1.9

                        0.5

                        0.0023

                        53

                        36

                        1.5

                        0.7

                        0.0106

                        agree

                        concordance

                        B

                        PLP2

                        294

                        157

                        1.9

                        0.5

                        0.0051

                        270

                        190

                        1.4

                        0.7

                        0.0151

                        agree

                        concordance

                        B

                        PAFAH1B1

                        181

                        98

                        1.9

                        0.5

                        0.0006

                        556

                        387

                        1.4

                        0.7

                        0.0089

                        disagree

                        concordance

                        B

                        TIMM22

                        42

                        23

                        1.8

                        0.5

                        0.0392

                        126

                        82

                        1.5

                        0.6

                        0.0001

                        disagree

                        concordance

                        B

                        AMOTL2

                        308

                        173

                        1.8

                        0.6

                        0.0015

                        776

                        484

                        1.6

                        0.6

                        0.0113

                        agree

                        concordance

                        B

                        ATP1B3

                        668

                        386

                        1.7

                        0.6

                        <0.0001

                        832

                        449

                        1.9

                        0.5

                        0.0015

                        disagree

                        concordance

                        C

                        DAPK1

                        181

                        117

                        1.5

                        0.6

                        >0.05

                        186

                        146

                        1.3

                        0.8

                        >0.05

                        agree

                        concordance

                        B

                        TFRC

                        894

                        606

                        1.5

                        0.7

                        0.0089

                        386

                        216

                        1.8

                        0.6

                        0.0062

                        agree

                        concordance

                        B

                        ATG3

                        200

                        139

                        1.4

                        0.7

                        0.0106

                        342

                        319

                        1.1

                        0.9

                        >0.05

                        agree

                        concordance

                        B

                        RNF7

                        177

                        125

                        1.4

                        0.7

                        0.0178

                        54

                        63

                        0.9

                        1.2

                        >0.05

                        agree

                        concordance

                        A

                        IL18

                        21

                        16

                        1.4

                        0.7

                        0.0148

                        125

                        104

                        1.2

                        0.8

                        0.0210

                        agree

                        concordance

                        C

                        CRKL

                        38

                        28

                        1.4

                        0.7

                        >0.05

                        18

                        23

                        0.8

                        1.3

                        >0.05

                        agree

                        concordance

                        B

                        XIST

                        103

                        76

                        1.4

                        0.7

                        >0.05

                        256

                        378

                        0.7

                        1.5

                        >0.05

                        agree

                        discordance

                        C

                        PI4KA

                        59

                        44

                        1.4

                        0.7

                        0.0127

                        110

                        113

                        1.0

                        1.0

                        >0.05

                        agree

                        concordance

                        D

                        MSH6

                        62

                        47

                        1.3

                        0.8

                        >0.05

                        227

                        519

                        0.4

                        2.3

                        0.0010

                        disagree

                        discordance

                        C

                        LZTR1

                        82

                        69

                        1.2

                        0.8

                        >0.05

                        81

                        74

                        1.1

                        0.9

                        >0.05

                        agree

                        concordance

                        D

                        MLH1

                        171

                        150

                        1.1

                        0.9

                        >0.05

                        143

                        150

                        1.0

                        1.0

                        >0.05

                        agree

                        concordance

                        C

                        MYC

                        151

                        142

                        1.1

                        0.9

                        >0.05

                        119

                        212

                        0.6

                        1.8

                        >0.05

                        agree

                        discordance

                        B

                        PCOLCE2

                        22

                        21

                        1.0

                        1.0

                        >0.05

                        39

                        39

                        1.0

                        1.0

                        >0.05

                        agree

                        concordance

                        C

                        CCND3

                        136

                        139

                        1.0

                        1.0

                        >0.05

                        101

                        134

                        0.7

                        1.3

                        0.0127

                        agree

                        concordance

                        D

                        KRAS

                        157

                        162

                        1.0

                        1.0

                        >0.05

                        150

                        200

                        0.8

                        1.3

                        >0.05

                        agree

                        concordance

                        A

                        SEPT9

                        880

                        918

                        1.0

                        1.0

                        >0.05

                        543

                        394

                        1.4

                        0.7

                        >0.05

                        agree

                        concordance

                        D

                        RB1

                        67

                        73

                        0.9

                        1.1

                        >0.05

                        166

                        225

                        0.7

                        1.4

                        >0.05

                        agree

                        concordance

                        D

                        BRCA2

                        10

                        12

                        0.8

                        1.2

                        >0.05

                        15

                        23

                        0.6

                        1.6

                        0.0210

                        agree

                        concordance

                        B

                        SNX4

                        43

                        52

                        0.8

                        1.2

                        >0.05

                        199

                        339

                        0.6

                        1.7

                        0.0042

                        agree

                        concordance

                        A

                        BTN3A2

                        40

                        48

                        0.8

                        1.2

                        >0.05

                        89

                        173

                        0.5

                        1.9

                        0.0005

                        disagree

                        concordance

                        C

                        TFF1

                        12

                        16

                        0.7

                        1.4

                        >0.05

                        226

                        61

                        3.7

                        0.3

                        <0.0001

                        disagree

                        discordance

                        B

                        NUP85

                        71

                        101

                        0.7

                        1.4

                        >0.05

                        85

                        134

                        0.6

                        1.6

                        0.0028

                        agree

                        concordance

                        C

                        JUND

                        759

                        1181

                        0.6

                        1.6

                        >0.05

                        1725

                        2479

                        0.7

                        1.4

                        >0.05

                        agree

                        concordance

                        B

                        OSBPL11

                        46

                        74

                        0.6

                        1.6

                        0.0151

                        56

                        148

                        0.4

                        2.6

                        <0.0001

                        disagree

                        concordance

                        D

                        BRCA1

                        15

                        24

                        0.6

                        1.6

                        >0.05

                        27

                        40

                        0.7

                        1.5

                        >0.05

                        agree

                        concordance

                        B

                        SR140

                        144

                        243

                        0.6

                        1.7

                        0.0089

                        13

                        64

                        0.2

                        5.0

                        <0.0001

                        disagree

                        concordance

                        D

                        BRAF

                        27

                        46

                        0.6

                        1.7

                        0.0089

                        22

                        47

                        0.5

                        2.1

                        <0.0001

                        disagree

                        concordance

                        C

                        ZNF74

                        12

                        21

                        0.6

                        1.8

                        0.0042

                        16

                        44

                        0.4

                        2.8

                        0.0002

                        disagree

                        concordance

                        B

                        TOMM70A

                        212

                        383

                        0.6

                        1.8

                        0.0004

                        115

                        306

                        0.4

                        2.7

                        <0.0001

                        agree

                        concordance

                        B

                        RPL24

                        1895

                        3503

                        0.5

                        1.8

                        0.0002

                        1834

                        4179

                        0.4

                        2.3

                        0.0003

                        agree

                        concordance

                        C

                        HSPD1

                        899

                        1682

                        0.5

                        1.9

                        0.0002

                        461

                        1189

                        0.4

                        2.6

                        0.0004

                        agree

                        concordance

                        D

                        MSH2

                        27

                        53

                        0.5

                        2.0

                        0.0023

                        112

                        495

                        0.2

                        4.4

                        <0.0001

                        disagree

                        concordance

                        B

                        MYNN

                        27

                        55

                        0.5

                        2.1

                        0.0001

                        16

                        40

                        0.4

                        2.5

                        0.0005

                        agree

                        concordance

                        D

                        ERBB2

                        99

                        230

                        0.4

                        2.3

                        0.0003

                        50

                        142

                        0.4

                        2.8

                        0.0002

                        agree

                        concordance

                        B

                        ICAM2

                        14

                        34

                        0.4

                        2.5

                        0.0011

                        13

                        25

                        0.5

                        1.9

                        0.0089

                        agree

                        concordance

                        B

                        CEP70

                        23

                        59

                        0.4

                        2.6

                        <0.0001

                        56

                        182

                        0.3

                        3.3

                        <0.0001

                        agree

                        concordance

                        B

                        TMEM97

                        70

                        195

                        0.4

                        2.8

                        0.0015

                        51

                        140

                        0.4

                        2.8

                        0.0004

                        disagree

                        concordance

                        B

                        CD300A

                        11

                        36

                        0.3

                        3.3

                        <0.0001

                        4

                        36

                        0.1

                        9.2

                        0.0006

                        agree

                        concordance

                        A

                        STAT1

                        30

                        109

                        0.3

                        3.6

                        0.0127

                        48

                        110

                        0.4

                        2.3

                        0.0210

                        agree

                        concordance

                        A

                        EVI1

                        11

                        197

                        0.06

                        17.5

                        <0.0001

                        36

                        636

                        0.06

                        17.5

                        <0.0001

                        agree

                        concordance

                        C

                        APOE

                        7

                        126

                        0.06

                        17.9

                        <0.0001

                        39

                        326

                        0.12

                        8.4

                        <0.0001

                        agree

                        concordance

                        A

                        CP

                        7

                        295

                        0.02

                        43.5

                        <0.0001

                        33

                        972

                        0.03

                        29.3

                        <0.0001

                        agree

                        concordance

                        A

                        RGS1

                        2

                        112

                        0.02

                        47.0

                        <0.0001

                        3

                        169

                        0.02

                        56.5

                        <0.0001

                        agree

                        concordance

                        A

                        SPON1

                        5

                        271

                        0.02

                        57.8

                        <0.0001

                        6

                        257

                        0.02

                        44.9

                        <0.0001

                        agree

                        concordance

                        A

                        CD24

                        6

                        481

                        0.01

                        77.2

                        <0.0001

                        63

                        3697

                        0.02

                        58.5

                        <0.0001

                        agree

                        concordance

                        A

                        IGKC

                        7

                        991

                        0.01

                        151.6

                        <0.0001

                        27

                        873

                        0.03

                        32.6

                        0.0008

                        agree

                        concordance

                        A

                        IGHG1

                        3

                        1262

                        0.003

                        374.3

                        <0.0001

                        19

                        203

                        0.10

                        10.5

                        <0.0001

                        agree

                        concordance

                        1See Table 1 for description of categories of selection criteria. 2Fold change >2 or <0.5 (bold) between NOSE (N) and TOV (T) gene expression comparison. 3Welch Rank Sum Test p<0.001 (italics) difference between NOSE (N) and TOV (T).

                        As shown in Figure 2A, there was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between TOV versus NOSE samples (R = 0.93) and by Bland-Altman analysis (Figure 2B), where the majority of probes exhibited expression profiles in comparative analyses that fell within the 95% limits of agreement. Both statistical methods of comparative analysis of log2 fold differences show minimal variance as the mean increases regardless of the direction of expression difference evaluated: genes selected based on over- or under-expression in TOV samples relative to NOSE samples. Although there were examples of expression differences which fell outside the 95% limits of agreement as observed in the Bland-Altman analysis such as for RGSF4, PDPN, IGKC, IGHG1, C1QTNF1, TFF1 and IL1B (Figure 2B), both the directionality and magnitude of TOV versus NOSE expression patterns were generally consistent (Figure 2A and Table 2).
                        http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-7-55/MediaObjects/12967_2009_Article_366_Fig2_HTML.jpg
                        Figure 2

                        Comparison of the fold change difference in expression between NOSE and TOV samples for the Ziplex and Affymetrix platforms. A: The log2 fold change between the NOSE and TOV samples (mean NOSE signal intensity/mean TOV signal intensity) was calculated for the expression values of all 93 probes and plotted. Linear regression was performed resulting in the following model: log2 Affymetrix NOSE/TOV = 0.180098 + 1.0251794 log2 Ziplex NOSE/TOV with a Pearson's correlation coefficient (R) of 0.93. Probes that were not significant (p > 0.001 based on a Welch Rank Sum test) on either platform are indicated in grey, probes significant (p < 0.001 based on a Welch Rank Sum test) on both platforms are indicated in black, on only the Ziplex platform are indicated in blue and on only the Affymetrix platform in green. B: Bland-Altman plots for expression values of all probes. Values determined to be outliers are indicated in the mean-difference (of the log2 fold change values) plot. A difference in log2 fold change of 0 is indicated by a solid black line. The upper and lower 95% limits of agreement for the difference in log2 fold change are indicated by red dashed lines, and arrows on the right hand side. Expression values that fall outside of these lines are considered outliers and are identified by their gene name.

                        Discussion

                        The Ziplex array technology as applied to ovarian cancer research was capable of reproducing expression profiles of genes selected based on their Affymetrix GeneChip patterns. A high concordance of gene expression patterns was evident based on overall correlations, significance testing and fold-change comparisons derived from both platforms. The Ziplex array technology was validated by testing the expression of genes exhibiting not only significant differences in expression between normal tissues (NOSE) and ovarian cancer (TOV) samples but also the vast range in expression values exhibited by these samples using the Affymetrix microarray technology. Notable also is that comparisons were made between Affymetrix GeneChip data that was derived using MAS5 software rather than RMA analysis. We have routinely used MAS5 derived data in order to avoid potential skewing of low and high expression values which could occur with RMA treated data sets as this is more amenable to data sets of limited sample size [6, 23, 25, 26, 30]. MAS5 derived data also allows for exclusion of data that may represent ambiguous expression values as reflected in a reliability score based on comparison of hybridization to sets of probes representing matched and mismatch sequences complementary to the intended target RNA sequence. A recent study has re-evaluated the merits of using MAS5 data with detection call algorithms demonstrating its overall utility [31]. Our results are consistent with a previous study which had tested the analytical sensitivity, repeatability and differential expression of the Ziplex technology within a MAQC study framework [21]. As with all gene expression platforms, reproducibility is more variable within very low range of gene expression. Gene expression values in the low range across comparable groups would unlikely be developed as RNA expression biomarkers at the present time regardless of platform used. The MAQC study included a comparison of Xceed Molecular platform performance with at least three major gene expression platforms in current use in the research community, such as Affymetrix GeneChips, Agilent cDNA arrays, and real-time RT-PCR. The implementation of some of these various technology platforms in a clinical setting may require significant infrastructure which may be awkward to implement due to the level of expertise involved. In some cases, costs may also be prohibitive but this should diminish over time with increase in usage in clinical settings. It is also not clear that expression biomarkers are readily adaptable to all cancer types as this requires sufficient clinical specimens to extract amounts of good quality RNA for RNA biomarker screening to succeed. Tumor heterogeneity is also an issue. The large size and largely tumor cell composition of ovarian cancer specimens may render this disease more readily amenable to the development and implementation of RNA biomarker screening strategies in order to improve health care of ovarian cancer patients. The ease with which to use the Ziplex Automated Workstation focus array and the fact that it appears to perform overall as well as highly sensitive gene expression technologies including real-time RT-PCR, suggests that this new platform might be amenable to translational research of gene expression-based biomarkers for ovarian cancer initially identified from established large-scale gene expression platforms.

                        Data normalization of gene expression values is a subject of intense study and is a major consideration when moving from one technology platform to another [4, 5]. In this study, data normalization of the Ziplex data was achieved by using the expression values derived from seven genes, each of which had low CV values across all samples tested. Since the input quantity of amplified RNA was equivalent for all Ziplex arrays, raw data could also have been used in our analysis. A statistical analysis based on correlations and fold-changes found negligible differences between raw and normalized data (not shown). Affymetrix GeneChip and Ziplex systems also differ in a number of technical ways that may affect the determination of gene expression. Affymetrix probe design is based on 11 oligonucleotide probes, 25 base pairs in size, within a target sequence of several hundred base pairs. The gene expression value is based on the median of the measured signal from the 11 probes. The probe design for the Ziplex system is based on oligonucleotide probes ranging from 35 to 50 bases. In this study three probes were designed and tested for each target gene and a single optimal probe was chosen. The visualization system for gene expression differs for both platforms where expression using the Ziplex array is measured by chemiluminescence, whereas fluorescence is used for the Affymetrix GeneChip. In spite of these differences, our findings along with an independent assessment of the Ziplex system [21] indicated a high degree of correspondence in expression profiles generated across both platforms. The overall findings are not surprising given that the probe design was intentionally targeted to similar 3'UTR sequences for the tested gene. Thus, the overall reproducibility of expression profiles along with the possibility of using raw data would be an attractive feature of applying the Ziplex system to validated biomarkers that were discovered using the Affymetrix platform.

                        The expression patterns of many of the tested genes were previously validated by an independent technique from our research group. RT-PCR analyses of ovarian cancer samples validated gene expression profiles of TMEM158, GBE1 and HEG1 from a chromosome 3 transcriptome analysis [25] and IGFBP4, PTRF and C1QTNF1 from a chromosome 17 transcriptome analysis [26]. The Ziplex platform also revealed over-expression of genes (ZNF74, PIK4CA, SERPIND1, LZTR1 and CRKL) associated with a chromosome 22q11 amplicon found in the OV90 EOC cell line and initially characterized by earlier generation Affymetrix expression microarrays and validated by RT-PCR and Northern blot analysis [23]. Differential expression of SPARC, a tumor suppressor gene implicated in ovarian cancer, has been shown to give consistent expression profiles in EOC cell lines and samples across a number of Affymetrix GeneChip® platforms and by RT-PCR from our group and others [6, 30, 32]. This indicates the utility of using older generation Affymetrix GeneChip data where good concordance can be observed with historical data and the accuracy of the earlier generation GeneChips has been evaluated by alternative techniques in the literature [6, 23]. This is an important consideration particularly given the large number of historical data sets that are available for further mining of potential gene expression biomarkers. Northern blot analysis has validated expression of MYC, HSPD1, TP53 and PGAM1 which were initially found to be differentially expressed in our EOC cell lines by the prototype Affymetrix GeneChip [6]. Concordance of gene expression was also evident from the 10 genes (see Table 1) selected based on an Affymetrix U133A microarray analysis of TOV samples and short term cultures of NOSE samples reported by an independent group [3]. BTF4 is a potential prognostic marker for ovarian cancer and was originally identified by Affymetrix microarray technology and then validated by real-time RT-PCR analysis [14]. Assaying the expression of BTF4 in clinical specimens is of particular interest because at the time of study there was no available antibody, illustrating the need for a reliable and accurate quantitative gene expression platform for RNA molecular markers.

                        Conclusion

                        It is becoming increasingly apparent that expression signatures involving multiple genes can be correlated with various clinical parameters of disease, and in turn that these signatures could be used as biomarkers [4, 5]. Although the expression signatures are gleaned from the statistical analyses of transcriptomes from genome-wide expression analyses, such as with use of Affymetrix GeneChip, the use of such arrays requires technical expertise and infrastructure that is not at the present time readily adaptable to clinical laboratories. In this study we have shown the concordance of the expression signatures derived from Affymetrix microarray analysis by the Ziplex array technology, suggesting that it is amenable for translational research of expression signature biomarkers for ovarian cancer.

                        List of abbreviations used

                        RNA: 

                        ribonucleic acid

                        mRNA: 

                        messenger ribonucleic acid

                        UTR: 

                        untranslated region

                        R: 

                        correlation coefficient

                        MAQC: 

                        MicroArray Quality Control

                        RT-PCR: 

                        reverse transcription polymerase chain reaction

                        NOSE cells: 

                        normal ovarian surface epithelial cells

                        TOV: 

                        ovarian tumor

                        EOC: 

                        epithelial ovarian cancer

                        BLAST: 

                        Basic Local Alignment Search Tool

                        NCBI: 

                        National Centre for Biotechnology Information

                        RIN: 

                        RNA integrity number

                        HRP: 

                        horseradish peroxidase

                        SNR: 

                        signal to noise ratio

                        SI: 

                        signal intensity.

                        Declarations

                        Acknowledgements

                        Manon Deladurantaye provided technical assistance with sample preparation. PT is an Associate Professor and Medical Scientist at The Research Institute of the McGill University Health Centre which receives support from the Fonds de la Recherche en Santé du Québec (FRSQ). AB is a recipient of a graduate scholarship from the Department of Medicine and the Research Institute of the McGill University Health Centre and PW is a recipient of a Canadian Institutes of Health Research doctoral research award. The ovarian tumor banking was supported by the Banque de tissus et de données of the Réseau de recherche sur le cancer of the FRSQ affiliated with the Canadian Tumour Respository Network (CRTNet). This work was supported by grants from the Genome Canada/Génome Québec, the Canadian Institutes of Health Research and joint funding from The Terry Fox Research Institute and Canadian Partnership Against Cancer Corporation (Project: 2008-03T) to PT, AMMM and DP.

                        Authors’ Affiliations

                        (1)
                        Department of Human Genetics, McGill University
                        (2)
                        Xceed Molecular
                        (3)
                        The Research Institute of the McGill University Health Centre
                        (4)
                        Centre de Recherche du Centre hospitalier de l'Université de Montréal/Institut du cancer de Montréal
                        (5)
                        Département de Médicine, Université de Montréal
                        (6)
                        Département de Obstétrique et Gynecologie, Division of Gynecologic Oncology, Université de Montréal
                        (7)
                        Department of Medicine, McGill University

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                        Copyright

                        © Quinn et al. 2009

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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