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.

Takara Bio's One Step PrimeScript III RT-qPCR Kit allows cDNA synthesis from RNA using PrimeScript Reverse Transcriptase, followed by PCR amplification with Takara Ex Taq Hot Start Version in a single, uninterrupted procedure. PCR amplification products are detected and monitored in real time with either probe- or TB Green-based detection.

One-step RT-qPCR kits that utilize probe-based detection must be accurate, specific and reproducible. Our One Step PrimeScript RT-PCR Kit (Perfect Real Time) passes these requirements with flying colors. This kit is available in both 100 and 500 reaction sizes.

Oligonucleotides modified with a fluorophore (e.g., FAM) at the 5' end and quencher (e.g., TAMRA) at the 3' end are included in the reaction. During the annealing step, the probe specifically hybridizes to the template DNA and the fluorophore's fluorescence is suppressed by the quencher. During the extension step, the 5'→3' exonuclease activity of Taq DNA polymerase degrades the probe hybridized to the template. This prevents quenching and allows fluorescence emission. The amount of amplified product can be monitored by measuring the fluorescence intensity.

One-step RT-qPCR kits that utilize TB Green-based detection are rapid, efficient, and sensitive. When you want rapid and cost-effective results in a single-tube protocol, utilize our One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time). This kit is available in both 100 and 500 reaction sizes.

This method detects fluorescence produced during the amplification process by adding a DNA intercalating dye (TB Green) that fluoresces upon binding to double-stranded DNA. Following the synthesis and binding of TB Green to DNA synthesized during RT-PCR, the quantity of amplified DNA and the melting point of the resulting amplicon can be measured.

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

Ma, W. et al. Zika virus causes testis damage and leads to male infertility in mice. Cell 167, 1511–1524.e10 (2016).

Cat. # RR064A was used to sensitively detect Zika viral RNA levels from multiple tissues in Zika virus-infected mice. These data were able to discern higher viral levels in testis, associated with infertility.

Xie, Y., Wang, M., Xu, D., Li, R. & Zhou, G. Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR. J. Virol. Methods 162, 64–68 (2009).

Cat. # RR064A was used in the development of a one-step quadruplex RT-PCR method for detecting viruses in sugarcane. This rapid and sensitive technique greatly reduced cost and labor since multiple infections could be tested for in one sample.

Zhang, N. et al. Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR. Virol. J. 8, 374 (2011).

Cat. # RR086A was used to develop a specific, sensitive, and reproducible assay for bovine viral diarrhea virus, a surrogate model for hepatitis C virus. The assay was 10-fold more sensitive than conventional assays and showed no primer dimers or nonspecific products.

Zou, Q. et al. Use of Praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice. PLoS One 7, e34865 (2012).

Cat. # RR086A was used to measure H5N1 infection levels in mouse lung tissue. This enabled the testing of a novel adjuvant, PZQ, which was able to reduce virus loads and prolong survival.