Blood is a critical tissue for the understanding of disease and the development of disease treatments. It is a ubiquitous tissue that interacts throughout the body and literally acts as a sensor of physiological conditions [1, 2]. While many assays exist to extract this critical knowledge from blood for proteins, lipids and single genes, development of genome-based biomarker assays has been a challenge. This is due to the high and variable levels of globin transcripts that interfere with achieving significant sensitivity . To this end, several commercial solutions have been developed to prevent the generation of globin transcripts during sample preparation. We and others have shown that many of these methods do improve data quality (figure 1; [14, 18, 22]). However, using Ribo-SPIA amplification, we have demonstrated that the globin transcript can be fully represented in the target and its effect on hybridization data can be ameliorated through the highly-specific properties of DNA:DNA binding.
Most microarray platforms typically utilize a fixed probe length of DNA, whether spotted in place or synthesized in situ. This critical fact defines much of the performance of the microarray in terms of sensitivity (DNA will allow a certain amount of promiscuous cross hybridization effecting background determination) and specificity (the fidelity of hybridization between the probe and the target). Standard amplification techniques rely on the RT/IVT method developed by Eberwine and Van Gelder . This method amplifies mRNA and incorporates the necessary label using an in vitro transcription step that is robust and efficient. The amplified material produced is a cRNA whose characteristics for sensitivity and specificity are acceptable, but not as good as a DNA target. DNA has been shown to perform better as a hybridization target than RNA, since it is highly specific and less susceptible to cross-hybridization . These characteristics also support the use of DNA as a means of mitigating the effects of globin, and potentially other highly abundant interfering transcripts.
Evaluation of the hybridization characteristics show that cDNA probes generated by Ribo-SPIA amplification perform better than using the standard cRNA method of amplification and labeling. cDNA hybridizations have greater intensity (low Scale Factor) and better discrimination between true signal and background (measured as a higher percentage of present calls) (additional file 2). Not only are there improvements in hybridization metrics, but the deleterious effects of globin cross-hybridization are reduced. As seen in figures 1 and 5, and quantified in table 1, the correlation between the amount of globin in a sample and the number of false positive signatures is greatly reduced when either globin mitigation strategy is used. However, we found that the Ribo-SPIA method significantly outperformed the PNA method. Indeed, there is an improved detection sensitivity of nearly 4-fold, a reduction of the globin artifact by 5-fold and an increase in statistical power (signal to noise) of more than 3-fold. The loss of correlation between the amount of globin in the sample and the number of false detections indicates the benefits of this approach. This improved performance was consistent whether the background sample was of either a cell line or whole blood origin.
Concomitant with a reduced correlation between globin and false positive signatures is an increase in the number of true signatures detected. Irrespective of globin interference, it is useful to measure the sensitivity of all methods. When comparing the spiked-in liver vs brain signatures, the Ribo-SPIA protocol identified 4,000 more significant genes than the standard no treatment control. PNA had little to no effect in sensitivity with an increase of less than 200 genes (table 1). This benefit is magnified in the presence of cross-contaminating globin. Figures 2 and 5 show the benefits of globin mitigation. Both the Ribo-SPIA and PNA methods increase the number of true detections (as measured by the number of brain or liver signatures detected) when compared to no treatment. As before, the Ribo-SPIA protocol is far superior to the standard PNA protocol. Figure 6 shows a ROC-like analysis where genes associated with globin amount are considered false positives and the total number of signatures detected is derived by building a ratio between the Jurkat spiked with brain and Jurkat spiked with liver. This presentation shows that for a given level of false positives attributable to globin cross-hybridization, both the Ribo-SPIA and PNA protocols are more sensitive than a no treatment control, with the Ribo-SPIA significantly outperforming PNA.
During the preparation of this manuscript, a number of other teams have published studies evaluating methodologies for whole genome expression profiling from whole blood. Many of these used similar methodologies for objectively measuring expression profiling performance [14, 22]. Others have noted the benefits of usincDNA targets for profiling, although in some cases it was noted that earlier versions of the Ribo-SPIA protocol were used . It should be noted that we used the NuGEN Ovation WB kit, which is an improved method over early versions.
Other recent work has compared profiling using cDNA to other methods, including direct isolation of PBMCs . In any research, the cause of negative results is often unknown and dismissed based on several reasons. For example, it was reported that robust transcriptional signature of acute graft rejection in tissue biopsies could not be detected in whole blood even after using cDNA-based amplification and hybridization . The cause is unknown and could be due to the biological relevance of whole blood in detection of graft rejection or inability to fully mitigate globin effects.
There are several examples in the literature of ex vivo gene expression profiling as well as experiments looking at the SAHA-induced expression profiling [31, 33–37]. The latter generally rely on the isolation of PBMCs in order to mitigate globin contamination. This extra processing can induce signatures of its own and thus reduce sensitivity [10, 12, 38, 39]. A significant benefit of the NuGEN Ovation WB protocol is that such extra manipulation is not necessary and pre-amplification noise is not introduced. The goal of the study was to demonstrate the utility of cDNA targets for whole blood gene profiling. Using a cDNA target derived from the Ribo-SPIA protocol, the number of genes correlated to globin input was reduced by 5-fold compared to a no treatment control, with a 4-fold increase in tissue-specific genes. Although the study was not specifically designed or powered to identify new clinically-relevant biomarkers, it was designed to capture the time-and dose-dependent biological response of whole blood to SAHA administration. These data support the concept that cDNA hybridization to microarrays is a valuable methodology for identifying clinically-relevant gene expression patterns in whole blood and reveal previously obscured biomarkers.