FAQs qPCRBIO cDNA Synthesis Kit – Witec AG

Can miRNA be amplified using qPCRBIO cDNA Synthesis Kit?

miRNA can’t be amplified using qPCRBIO cDNA Synthesis Kit. UltraScript Reverse Transcriptase or UltraScript 2.0 Reverse Transcriptase should be used because they come with a buffer that does not contain random hexamers and oligod(T)s, which could interfere with miRNA specific primers.

Can qPCRBIO cDNA Synthesis Kit be used for endpoint and qPCR?

All our RTases and kits can be used for both endpoint and qPCR applications.

Can qPCRBIO cDNA Synthesis Kit reverse transcribe eukaryotic and prokaryotic RNA?

qPCRBIO cDNA Synthesis Kit can amplify both eukaryotic and prokaryotic RNA. Our mix contains random hexamers that are universal primers for all types of RNA, provided it contains sufficiently long fragments. Due to the presence of hexamers, RNA from all organisms can be reverse transcribed if it is available for the RTase. There are also oligo-d(T) included in the mix, which work with A-tailed RNA such as eukaryotic mRNA.

For a more selective kit use UltraScript Reverse Transcriptase or UltraScript 2.0 Reverse Transcriptase as the buffer for these products come without any hexamers and oligo(dT)s. This allows you to determine your own priming strategy.

Is a thermocycler required to perform reverse transcription?

Reverse transcription is an isothermal process so any instrument that can maintain stable temperature will be fine. We do not advise using water baths due to contamination. A thermocycler has the added advantage of a heated lid that circumvents the problem of tube cap condensation.

What is the dynamic range of qPCRBIO cDNA Synthesis Kit?

qPCRBIO cDNA Synthesis Kit can transcribe as little as 4pg and up to 0.4μg of RNA. The performance depends on the type of RNA, RNA quality and whether specific primers, oligod(Ts) or random hexamers are used. For higher capacity applications please use UltraScript 2.0 Reverse Transcriptase and cDNA Synthesis Kits.

What is the error rate of the RTase in qPCRBIO cDNA Synthesis Kit?

Like the great majority of the RTases available on the market UltraScript Reverse Transcriptase, as well as all other RTases we offer, are derived from wild-type Moloney Murine Leukemia Virus (MMLV) RTase and have an error rate of 1×10-4 errors/bp1. This also applies to RTases derived from Avian Myeloblastosis Virus (AMV).

Running the reaction at a higher temperature increases fidelity because it destabilises mismatched base pairs2. If low fidelity is required adding manganese will make RTases highly mutagenic and could increase their speed3.

1  Yasukawa, K. et al. Next-generation sequencing-based analysis of reverse transcriptase fidelity. Biochem Biophys Res Commun 492, 147-153, doi:10.1016/j.bbrc.2017.07.169 (2017).

2  Malboeuf, C. M., Isaacs, S. J., Tran, N. H. & Kim, B. Thermal effects on reverse transcription: improvement of accuracy and processivity in cDNA synthesis. Biotechniques 30, 1074-1078, 1080, 1082, passim, doi:10.2144/01305rr06 (2001).

3  Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl 2, 28-33 (1992).

What is the maximum length of product possible using qPCRBIO cDNA Synthesis Kit?

The RTase included in qPCRBIO CDNA Synthesis Kit can transcribe very long targets of up to 20 kB with sufficient optimisation1. However, the kit contains random hexamers that will align in random places along the sequence and this will greatly reduce the maximum length of a continuous product. Most of the products will be discontinuous to a large extent nonetheless, the whole sequence of RNA will be covered and subsequently be quantifiable with qPCR. This applies to any cDNA synthesis kit that incorporates random hexamers. If full-length continuous products are required, we recommend UltraScript Reverse Transcriptase or UltraScript 2.0 Reverse Transcriptase as these products come with a buffer without any hexamers and oligo(dT)s.

1  Thiel, V. et al. Effective amplification of 20-kb DNA by reverse transcription PCR. Anal Biochem 252, 62-70, doi:10.1006/abio.1997.2307 (1997).

What troubleshooting is there for low cDNA yield?

There could be multiple reasons for low yield occurring:

• The amount or the integrity of your RNA could be insufficient to reach a significant amount of product required for the subsequent endpoint or qPCR step. Try increasing the amount RNA and/or check the quality of your RNA using gel electrophoresis and RIN value. Consider an alternative purification protocol for your RNA and/or use RNase inhibitor early on. All our RTases contain RNase inhibitor but for some applications it might be necessary to add it before RT step
• You RNA prep may contain inhibitors such as haem, high concentrations of NaCl, SDS, guanidine thiocyanate, melanin, or calcium1. Try an alternative method of purification that gets rid of all substances that can inhibit reaction. You could also try diluting your RNA before the reaction. This will decrease the theoretical yield, but it will also dilute inhibitors to levels more permissible for the RTase to work efficiently. This could still provide enough product for subsequent steps.
• Some RNAs are inherently difficult to transcribe due to secondary structures and/or stretches of sequences that are not an ideal substrate for RTase. Try increasing the temperature and/or performing RNA denaturation/primer annealing, at 70°C for 10 minutes, before adding RTase.
• Using RNAseH treatment after RT step can increase the yield, especially for GC rich targets because RNA-DNA heteroduplexes are more stable than DNA-DNA duplexes.
• If your sequences are still underrepresented it might be necessary to use a more targeted approach with specific primers or just oligo(dT)s. We recommend UltraScript Reverse Transcriptase or UltraScript 2.0 Reverse Transcriptase that come with a buffer without any hexamers and oligo(dT)s for that purpose.

  1  Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors – occurrence, properties and removal. J Appl Microbiol 113, 1014-1026, doi:10.1111/j.1365-2672.2012.05384.x (2012).

Why is there is a false positive in the RT-qPCR analysis?

Your RNA sample might be contaminated with DNA. We suggest re-purifying your RNA using a better protocol or use thermolabile DNase to get rid of any contamination.