Hello, I'd like to ask whether it is necessary to add primers or oligo(dt) to Reverse transcription mastermix (MMLV RT)? Is it possible to mix Primer independent RT reaction? If you have any experience with reaction conditions for MMLV I would be grateful for All advices. Thank you.
Bohuš
Reverse transcription requires priming from a double-stranded region of nucleic acid, just like PCR does. If you prepare a reverse transcription reaction without any primers, you will get some cDNA, but it will not be an accurate or even representation of what is in your sample, nor will it be consistent between samples - the cDNA will be produced from small random RNA fragments acting as primers.
What are you trying to achieve? Why do you not want to use primers? If we know the goal of the experiment, maybe we can help :)
Hi, as Laura said, you need a primer(s) for good quality reverse transcription.
If you want to RT a range of transcripts then you can use random hexamers or random decamers. This has worked well for me when preparing cDNA for gene expression analysis. SuperScript (https://www.thermofisher.com/order/catalog/product/11754050) is MMLV based and may be helpful.
Alternatively you can make the reaction more targeted by using a feature- or gene-specific primer:
- For example, templates for 3' RACE can be generated using a primer with an oligo-dT region to target the polyA tail of mRNAs.
- Or you can use a gene-specific primer to target a specific gene/transcript.
In my experience, the latter is best for lowly abundant transcripts and depending on the gene of interest you may need to try many different primers.
- Badges
- Science method