PCRBIO 1-Step Go RT-PCR Kit PCRBIO 1-Step Go RT-PCR Kit PCRBIO 1-Step Go RT-PCR Kit PCRBIO 1-Step Go RT-PCR Kit PCRBIO 1-Step Go RT-PCR Kit

PCRBIO 1-Step Go RT-PCR Kit

PCRBIO 1-Step Go RT-PCR Kit is a convenient, easy-to-use kit for rapid and efficient cDNA synthesis as well as PCR in a single tube. The advanced buffer system, reverse transcriptase enzyme, and hot-start polymerase provide highly sensitive and specific 1-step RT-PCR from any RNA template including mRNA, synthetic RNA, and viral RNA sequences. A red mix format with gel tracking dye is now also available for sample visualization and direct loading onto agarose gel. Characteristics Thermostable reverse transcriptase enzyme RTase Go Wide temperature range from 45 °C to 55 °C Advanced RNase inhibitor Antibody-mediated hot-start technology for exceptional low-copy template detection High PCR yield under standard and fast PCR conditions Efficient specific amplification from complex samples including GC and AT-rich sequences Red mix with tracking dye available for direct gel loading
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MS CATProductSizePrice
PB10.53-05PCRBIO 1-Step Go RT-PCR Kit50 x 50 μL ReactionsContact us
PB10.53-10PCRBIO 1-Step Go RT-PCR Kit100 x 50 μL ReactionsContact us
PB10.53-50PCRBIO 1-Step Go RT-PCR Kit500 x 50 μL ReactionsContact us
PB10.55-05PCRBIO 1-Step Go RT-PCR Kit Red50 x 50 μL ReactionsContact us
PB10.55-10PCRBIO 1-Step Go RT-PCR Kit Red100 x 50 μL ReactionsContact us
PB10.55-50PCRBIO 1-Step Go RT-PCR Kit Red500 x 50 μL ReactionsContact us

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Technical Specifications

PCRBIO 1-Step Go RT-PCR Kit

Components 50 Reactions 100 Reactions 500 Reactions
20x RTase Go with RNase Inhibitor 1 x 125 μL 2 x 125 μL 10 x 125 μL
2x PCRBIO 1-Step Go Mix 1 x 1.25 mL 2 x 1.25 mL 10 x 1.25 mL

PCRBIO 1-Step Go RT-PCR Kit Red

Components 50 Reactions 100 Reactions 500 Reactions
20x RTase Go with RNase Inhibitor 1 x 125 μL 2 x 125 μL 10 x 125 μL
2x PCRBIO 1-Step Go Mix Red 1 x 1.25 mL 2 x 1.25 mL 10 x 1.25 mL

Reaction Information

Reaction Volume Storage
50 μL Upon receipt, the product should be stored between -30 to -20 °C.
If stored properly, the kit will retain full activity until the expiration date indicated.

Frequently Asked Questions (FAQs)

We recommend using a minimum of 2 minutes to activate the enzyme. Longer times, up to 15 minutes, can also be used without adversely affecting the enzyme.

One-step

Both cDNA synthesis and PCR occur in the same mix. This option is suitable for high-throughput applications due to its speed and ease of setup. It also reduces the risk of contamination. However, it is not ideal for poor quality RNA samples or if cDNA is required to be stored or analyzed separately.

Two-step

The cDNA synthesis and PCR reactions occur separately. This option is better suited if the cDNA product needs to be retained for analysis. It also allows for a higher level of reaction optimization. It permits control over the type and concentration of enzyme, RNA input, and cDNA concentration, leading to higher sensitivity compared to the one-step format.

No, RTase Go already contains an RNase inhibitor to prevent any breakdown and enhance sensitivity.

If you observe unusual late Ct values, try diluting the sample RNA. By doing this, you are diluting any inhibitors that may be present to concentrations where they do not inhibit the reaction. Additionally, try increasing the reverse transcription step to 55 °C and increasing the annealing/extension temperature. This can help resolve difficulties due to secondary structures present in the RNA samples and/or primers.

When inhibitor-related reaction inhibition is suspected, try reducing the sample amount1 or adding 0.4 – 4.4 mg/ml BSA to the reaction2.

To resolve more specific issues, please contact technical@pcrbio.com with the following information:

Amplicon size

Reaction setup

Cycling conditions

Screenshots of amplification plots and melt curves

 

1  Scipioni et al. A SYBR Green one-step real-time RT-PCR assay to detect human and bovine noroviruses and control for inhibition. Virology Journal.5:94 (2008). doi: 10.1186/1743-422X-5-94

2  Plante et al. The use of bovine serum albumin to enhance the detection of foodborne viruses washed from vegetable surfaces. Applied Microbiology. 52:3 (2010) doi: https://doi.org/10.1111/j.1472-765X.2010.02989.x

 

mRNA isolation is usually not necessary. The PCRBIO 1-Step Go RT-PCR Kit has been developed to work on samples containing as little as 1 pg of total RNA or 0.01 pg of mRNA.

If working with rare mRNA species, use a primer specific to the sequence in the RT reaction to enhance sensitivity.

Gene-specific primers can be used in the one-step reaction.

The PCRBIO 1-Step Go RT-PCR Kit has been developed to amplify amplicons up to 3 kb from eukaryotic DNA.

The PCRBIO 1-Step Go RT-PCR Kit has been developed for endpoint RT-PCR. The qPCRBIO Probe 1-Step Go and qPCRBIO SyGreen 1-Step Detect|Go are probe-based and dye-based options for real-time RT-PCR, respectively.

Yes, the PCRBIO 1-Step Go RT-PCR Kit can be used for small RNA samples. While we do not sell dedicated kits, all our RTases can be used for miRNA quantification and analysis.

We recommend using one of the following approaches:

  • Use of a universal RT primer and the addition of a poly(A) or poly(U) tail (e.g., by poly(U)-polymerase), followed by cDNA synthesis using universal primers1,2.
  • Use a gene-specific RT primer and skip the tailing step1,3-5.

If you are not familiar with the details of these approaches, please refer to the references listed below, which serve as guides.

1  Dave, V. P. et al. MicroRNA amplification and detection technologies: opportunities and challenges for point-of-care diagnostics. Lab Invest 99, 452-469, doi:10.1038/s41374-018-0143-3 (2019).

2  Mei, Q. et al. An easy and specific approach for real-time quantification of microRNAs using modified RT primers. PLoS One 7, e46890, doi:10.1371/journal.pone.0046890 (2012).

3  Chen, C. et al. Real-time quantification of microRNAs by stem–loop RT-PCR. Nucleic Acids Res 33, e179, doi:10.1093/nar/gni178 (2005).

4  Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P. & Johnson, J. M. A simple, quantitative, PCR-based method for detecting low-level microRNAs. RNA 11, 1737-1744, doi:10.1261/rna.2148705 (2005).

5  Androvic, P., Valihrach, L., Elling, J., Sjoback, R. & Kubista, M. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res 45, e144, doi:10.1093/nar/gkx588 (2017).

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