Figure 1
The specificity, sensitivity, and reproducibility of qRT-PCR. The qRT-PCR was performed using a ten-fold dilution series (2.5 × 105 to 2.5 copies/reaction) of purified PCR product. Results shown are from a single representative experiment that was conducted three times. (A) The qRT-PCR successfully amplified the ten-fold dilution series of template (2.5 × 105 to 2.5 copies/reaction; from left to right). The non-template control (NTC) showed no amplification. The threshold was automatically set by Sequence Detector Systems version 1.2.2 software to synchronize among experiments. The threshold cycle (CT) was determined by the cycle number at which the change in the fluorescence of the reporter dye (delta Rn) crossed the threshold; (B) The standard curve was generated by plotting the CT vs the number of purified PCR product copies (Logcopies). The slope and correlation coefficient (R2) were -3.38 and 0.9986, respectively; (C) Melting curve analysis for the Vβ1 subgroup consist of single subgroup gene and showed a single peak at 78 oC; (D) Melting curve analysis for the Vβ17 subgroup consisting of three subgroup genes showed multiple peaks, consistent with the expected heterogeneity among amplified products.