I'm running reverse transcription experiments to amplify single cell RNA. I do lysis for the cells, RT and then pcr amplification.The library products I'm getting from cell samples are shorter than the ones I'm getting from reference RNA. When changing the ratio of RT reaction used for subsequent PCR, the products get even shorter when the RT/PCR is higher. Any suggestions why the cells give shorter yield?
The RT enzyme you are using is also a factor. Some enzymes are better for long transcipts than others. I had a problem similar to yours and I was able to fix it when I switched my Reverse Transcriptase to a RNAse H minus. I used ThermoFisher Maxima H Minus Reverse Transcriptase.
For a better answer, more information is required, but some considerations include: what are your PCR conditions? How long is the RNA being amplified? Is your extension time long enough? Could the reference RNA contain introns that the cells have spliced out? How much shorter is your amplified product than the reference RNA?
The RT enzyme you are using is also a factor. Some enzymes are better for long transcipts than others. I had a problem similar to yours and I was able to fix it when I switched my Reverse Transcriptase to a RNAse H minus. I used ThermoFisher Maxima H Minus Reverse Transcriptase.
l think that there are 2 reasons might related to ur situation. Firstly, your RNA isolation or cDNA synthesis might be contaminated RNAse, did u use the RNAse inhibitor during cDNA synthesis?, secondly you should have a positive control in PCR to make sure the condition was also fair with reference DNA seq such as a recombinant clone)
Above suggestions are all good explanations. You could try a gel extraction on your shorter bands and then send the product for sequencing and check what the product is? I.e. is it just a shorter fragment of your expected product, another fragment of RNA is being targeted or possible primer dimers etc.
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