Analysis of one-step and two-step real-time RT-PCR using SuperScript III

Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) is a commonly used technique to analyze gene expression. There has been little research conducted to test if SuperScript III quantitative one-step (reverse transcription carried out in the same tube as PCR) and two-step (reverse transcription carried out in a separate reaction) RT-PCR systems provide similar real-time results. In this study, real-time reactions were set up using the housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), beta2-microglobulin (B2M), and RNA polymerase 2 subunit A (PolR2A). Reaction efficiencies were determined by generating standard curves using total RNA isolated from human skeletal muscle and brain. Reaction efficiencies ranged from 97.7+/-0.9% to 99.4+/-1.8% for one-step and 98.0+/-0.2% to 102.6+/-1.3% for two-step RT-PCR (R2 values for all reactions>or=0.995). The sensitivities of one-step and two-step methods, as measured by cycle threshold values, were similar for GAPDH and B2M. However, for the lesser expressed PolR2A mRNA there was a 5 cycle lower threshold for one-step. In summary, both SuperScript III one-step and two-step methods yield reaction efficiencies close to 100% and produce similar, accurate, linear standard curves. However, using the one-step method with gene-specific priming may be more sensitive for quantification of certain genes such as PolR2A.

FIGURE 1

Real-time reaction run with β₂-microglobulin primer/probe and the SSIII one-step quantitative reverse transcription polymerase chain reaction kit. A: Real-time fluorescence using 1:10-fold dilutions of total RNA from skeletal muscle run in triplicate. B: The standard curve generated from the cycle thresholds of each of the dilutions and the equation of the line of best fit.