FAQs

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.