In case of bacterial total RNA, since all mRNA will not have poly A tail. I am using only random hexamer. Is it enough are should i add T tail oligomer also with the random hexamer.
And what is the most accurate method to confirm cDNA synthesis?
Thank you.
Hi Nithin
Random hexamer is sufficient still you can do both and then check with a specific qPCR primers for the both cDNAs.
You can check your cDNA with normal PCR with any bacterial specific primes or 16S primes, but during cDNA synthesis your RNA should be DNA free.
You can check your DNA free RNA by normal PCR with 16S or any primers, in case of clean RNA there should be no bands.
Hope will work.
Hi Nithin,
just to add to Abhijit answer, many people would argue that unless you want to selectively work with mRNA, using random hexamers is a better option because it allows the retro transcription of very long mRNAs that would not be retro transcribed due to length if the oligodT was used.
Hope this helps,
Ezequiel
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