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20 kb RNA reverse transcription can be achieved with all our RTases because RTases jump on and off their primed substrates. Secondary structures, unspecific binding and mis-priming can be the most common causes for the reaction ending prematurely.
We advise using specific primers or oligo(dT) and optimising each aspect of the reaction, for example, the different enzyme concentrations, primers, primer concentration, temperature and temperature cycling and time. Also, the subsequent PCR/qPCR step might need optimisation.
We recommend UltraScript™ 2.0 RTase for reverse transcribing long templates because it can sustain higher temperatures for longer, which may be necessary as the probability of secondary structures in RNA increases with its length.
We’ve included the following paper for your reference1.
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).
miRNA should not be amplified using UltraScript™ cDNA Synthesis Kit. UltraScript™ cDNA Synthesis Kit Separate Oligos, 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.
All our RTases are can be used for miRNA quantification and analysis. However, we do not sell any dedicated kits.
We advise that you use one of the two following approaches:
- Use universal RT primers and add poly(A) or poly(U) tails (e.g. by poly(U)-polymerase), followed by cDNA synthesis using universal primers1,2.
- Use specific RT primers and omit the tailing step1,3-5.
If you are unfamiliar with the specifics of those approaches, please refer to the reference list below, which serve as a guideline.
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. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach. 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. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. 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).
All our RTases and kits can be used for both endpoint and qPCR applications.
UltraScript™ DNA 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.
Yes, however we recommend comparing it against UltraScript™ 2.0 for your applications since it is better-suited to handle the various inhibitors present in blood. We also recommend optimisation by performing a titration of the amount of blood/plasma sample for RT reaction.
All our RTases can be used in template switching applications. We currently do not provide a full protocol or dedicated kit but we recommend referring to this list of articles for reference1-4.
1 Takada, S. & Mano, H. Profiling of microRNA expression by mRAP. Nat Protoc 2, 3136-3145, doi:10.1038/nprot.2007.457 (2007).
2 Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181, doi:10.1038/nprot.2014.006 (2014).
3 Moldovan, N. et al. Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus. Front Microbiol 8, 2708, doi:10.3389/fmicb.2017.02708 (2017).
4 Zajac, P., Islam, S., Hochgerner, H., Lonnerberg, P. & Linnarsson, S. Base preferences in non-templated nucleotide incorporation by MMLV-derived reverse transcriptases. PLoS One 8, e85270, doi:10.1371/journal.pone.0085270 (2013).
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.
Both products are derived from wild-type MMLV reverse transcriptase and contain multiple mutations that enhance their function. They are each excellent RTases in their own right. UltraScript™ 2.0 is more thermostable up to 65 °C and above, resistant to inhibitors and capable of reverse transcribing large quantities of RNA up to 3.5 μg. UltraScript™ 2.0 can be also used for applications requiring sensitivity due to a low amount of RNA present, however, this requires substantial optimisation on the users side using the current formulation and we recommend using UltraScript™ instead. UltraScript™ can be used with 1-step kits whereas UltraScript™ 2.0 should be used only as a part of the 2-step process (RT then PCR). UltraScript™ 2.0 is especially recommended for long and difficult templates that contain large amounts secondary structure. Both RTases can be used with cDNA synthesis kit with/or without hexamers and oligo-d(T).
UltraScript™ RTase can transcribe as little as 4 pg and up to 0.4 μg 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.
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).
The RTase included in UltraScript™ 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).
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).
What troubleshooting is there if smears or non-specific products are visible after using UltraScript™ RTase?
There could be multiple reasons for the appearance of smears or non-specific products after the RT reaction. The following points can be considered for troubleshooting:
- It’s important to establish if the problem relates to the reverse transcription step or PCR/qPCR step, provided the PCR/qPCR step is performed downstream from the reverse transcription step. Use proper controls and troubleshoot the PCR/qPCR reaction to exclude this from the list of potential steps causing the above problems.
- Smearing can indicate primer oligomerisation. Oligomerisation can be caused if the oligomers are badly designed and if the template concentration is low. This will result in reverse transcription of primer-dimers or off-target sequences. We advise redesigning primers, increasing reaction temperature, increasing the amount of template, decreasing primer concentration or shortening the reaction time. These considerations can also be used for non-specific bands.
- Primers may not be specific to your target sequence and may require redesign.
- Primers against highly repetitive sequences can cause smearing and redesigning your primers may have to be considered.
- Your RNA may be degraded. Try increasing the amount of RNA and/or check the quality of your RNA using gel electrophoresis and RIN value. Try an alternative purification protocol for your RNA and/or use RNase inhibitor early on. All our RTases contain RNA inhibitor but for some applications it might be necessary to add it before the RT step.
- Consider the standard precautions when handling RNA such as using gloves, positive displacement pipettes with aerosol barrier, etc.
- Ensure there is no DNA contamination.
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.