I have been reading several publications where reverse transcription was performed and I have seen that in many cases RNase H is used after reverse transcription and during second strand synthesis, and in many others RNase H is not used. Trying to understand why, I have read in Superscript III (Invitrogen) manual that amplification of some PCR targets (>1 kb) may require the removal of RNA complementary to the cDNA and RNase H should be used. In my personal case, I just want to reverse transcribe my RNA, make double stranded cDNA and build Illumina libraries. Do I need to treat with RNase H after RT? Thanks in advance for any help with this.
Best wishes
Niccolò
Perhaps it is recommended to use it in order to avoid mispriming with the residual RNA strands left after reverse transcription. Most of DNA oligos have high affinity with RNA, and even stronger, that with DNA matrix.
Well I you might be aware that the RNase H activity degrades RNA from RNA-DNA duplexes which aids synthesis of double-stranded DNA. While, with long mRNA templates, the RNA generally degrades prematurely resulting in truncated cDNA. So minimizing RNase H activity might just do the trick when aiming to produce long transcripts for cDNA .Well depending on the company you get your kit, there are reverse transcriptases which have an intrinsic RNase H domain that removes the RNA during cDNA strand synthesis. Varying kits have different RTs, and most of them lack the RNase H activity. So just have a look at your manual.
Cheers!
Thank you for the information regarding RNase h.
Can someone help me with the 3'RACE experiment? I am facing hurdles while performing 3'RACE to find out 3'UTR from an unknown genome sequence. It would be very helpful. Thank you
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