Quantitative PCR (qPCR) is a powerful technique for the accurate analysis of gene expression. When starting with RNA samples, one must first perform a reverse transcription (RT) step to generate cDNA for the subsequent qPCR reaction. One-step RT-qPCR streamlines this workflow by performing the RT step in the same tube as the qPCR reaction (Figure 1).
In general, one-step RT-qPCR is best suited for applications where speed and throughput are required. As the single-tube protocol is easy to set up and compatible with liquid handlers and/or automated systems it allows for less hands-on time, reduces pipetting errors, and minimizes contamination. One-step RT-qPCR is best applied to workflows comprised of many samples with few target genes.
However, one-step RT-qPCR has some limitations. Since both the RT and qPCR steps take place in the same tube the reaction conditions cannot be optimized separately, which can lead to lower yields and/or efficiency in either step. Another limitation is that all the generated cDNA is used up in the subsequent qPCR step, meaning that no stocks of cDNA can be banked for further validation or experimentation.
We offer one-step RT-qPCR kits supporting both TB Green- (our proprietary green intercalating dye) and probe-based chemistries to meet your experimental needs and give you the flexibility to run a wide variety of applications.