Quantitative PCR (qPCR) is a common, powerful technique for the accurate analysis of gene expression. When starting with RNA samples, you must first perform a reverse transcription (RT) step to generate cDNA for the subsequent qPCR reaction. Two-step RT-qPCR performs the RT step in one tube and the qPCR reaction in a separate tube (Figure 1).
Two-step RT-qPCR employs a two-tube workflow that makes it possible to optimize the RT and qPCR steps separately, allowing maximum flexibility, specificity, and efficiency. Two-step RT-qPCR works best with workflows analyzing many targets in fewer samples. Another advantage is that two-step RT-qPCR generates cDNA in a separate tube from the qPCR reaction, allowing for cDNA stocks to be banked for future use.
Two-step RT-qPCR has some limitations. In general, the protocols are more time-consuming and involve more pipetting steps. This introduces more variability and increases the risk of contamination. Additionally, while the ability to optimize both the RT and qPCR steps separately can improve sensitivity and efficiency, it comes with the drawback of having to optimize two reactions instead of one. Lastly, the two-tube protocol cannot be as easily adapted to automated, high-throughput workflows, necessitating more hands-on-time.
We offer two-step RT-qPCR kits in multiple formats for convenience, flexibility, and specificity. All kits can be combined with our probe- or TB Green- (our proprietary green intercalating dye) based qPCR kits to meet your experimental needs and give you the flexibility to run a wide variety of applications.