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.

Our two-step RT-qPCR kits are optimized for real-time PCR. Each contains PrimeScript Reverse Transcriptase, which has exceptionally strong strand displacement activity for optimal efficiency during cDNA synthesis. The PrimeScript reverse transcriptase is robust, versatile, and well-suited for applications requiring full-length cDNA synthesis. It enables reverse transcription of virtually any RNA template, including templates that are GC-rich and/or have high levels of secondary structure, making our two-step RT-qPCR kits ideal for both routine and challenging RT applications. PrimeScript RTase is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) enzyme, verified to be RNase H Minus, and is engineered to allow full-length cDNA synthesis reactions at lower temperatures (e.g., 42°C). This decreases the risk of RNA degradation that occurs during conventional reactions performed at higher temperatures.

We provide our two-step RT-qPCR kits in three formats to suit your experimental needs:

• For convenience, we have a complete master mix that includes primers and reverse transcriptase in a single tube—PrimeScript RT Master Mix (Perfect Real Time), Cat. No. RR036A/B
• For flexibility, we have a kit with primers and reverse transcriptase provided as separate components to let you optimize your reactions—PrimeScript RT Reagent Kit (Perfect Real Time), Cat. No. RR037A/B
• For maximum specificity, we have a kit that also includes a genomic DNA removal reagent—PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time), Cat. No. RR047A/B

Here are a few examples of research that's been driven using our two-step RT-qPCR kits:

Makino, K. et al. Inhibition of uterine sarcoma cell growth through suppression of endogenous tyrosine kinase B signaling. PLoS One 7, e41049 (2012).

Cat. No. RR037A was used to analyze gene expression levels in frozen and FFPE uterine leiomyosarcoma samples. The findings implicated TrkB signaling and provided potential drug targets for cancer therapies.

Mizutani, R. et al. Identification and characterization of novel genotoxic stress-inducible nuclear long noncoding RNAs in mammalian cells. PLoS One 7, e34949 (2012).

Cat. No. RR036A was used to identify a variety of novel lncRNAs using transcriptomic sequencing and bioinformatics.

Sato, Y., Inoue, M., Yoshizawa, T. & Yamagata, K. Moderate hypoxia induces β-cell dysfunction with HIF-1-independent gene expression changes. PLoS One 9, e114868 (2014).

Cat. No. RR047A was used to detect alterations of gene expression in primary islet cells under hypoxic conditions. These findings could implicate novel roles in β-cell dysfunction in type 2 diabetes.