FAQs
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FAQs
Browse our frequently asked technical questions by selecting a product below.
The cDNA yield is low. What can I do to help?
We advise you first ensure that cDNA yield is low rather than downstream PCR/qPCR yield or Cts are unsatisfactory. If the latter is the case, refer to “How can high Ct’s in the qPCR step or low PCR product on the gel after a RT step (PCR/qPCR problem) be alleviated?”. If the former is the case, there could be multiple reasons for low yield occurring when using UltraScript 2.0.:
- 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).
