Analysis of one-step and two-step real-time RT-PCR using SuperScript III.
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.
Wacker
Michael J
MJ
Applied Physiology Laboratory, 5059 Haworth Hall, 1200 Sunnyside Ave, University of Kansas, Lawrence, KS 66045, USA. mjwacker@ku.edu
Godard
Michael P
MP
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D001613:beta 2-Microglobulin / Q000235:genetics
D001613:beta 2-Microglobulin; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012319:RNA Polymerase II