Hi!
A special primer has to be used with only 19 bp poly dT (Tm 44.9) instead of 30 bp in original study (Tm 55.2). Will this induce failure of mRNA capture? Will this cause RNA detachment in Reverse transcription using Superscript II (@42 ℃)? If this is a problem, how about start reverse transcription @ 37℃ or 40℃ for 20min, then rise it to 42℃？ Will superscript II be completely block at lower temperature?
You can quite happily perform reverse transcription using random hexamers or nonamers, which have TMs well below 40 degrees. For this, it is typical to incubate the reactions at room temp for ~10 minutes, followed by 20-25mins at 42 (or whatever your RTase usually prefers). The initial low temp allows annealing and enough primer extension to keep everything bound for subsequent additional reverse transcription.
So, yes: an initial low temp incubation should allow decent efficiency.
You could also consider using oligodT-VN (19 Ts, then C, G or A, then any base): this will only bind to polyA tails immediately proximal to your 3' UTRs, maximising your chances of capturing actual mRNA sequence.
Very helpful answer, Thank you! John Hildyard
To confirm, I load the reagents and enzyme (superscript ii) before 10 min room temperature incubation is it?
Also, we do have 19T-V 3‘, yet not 19T-VN (still limit by length, there are certain index in front of poly T). Will this somehow helps?
Thanks again!
19T-V is better than 19T in targeting the beginning of the polyA site, yes. I believe 19T-VN just gives a slightly tighter clamp.
As to the reaction conditions, yes: heat your RNA + oligodT to 65 for 5 mins, crash cool on ice, then add the buffer/dNTPs/enzyme, mix well, spin down briefly, leave at RT for 10mins, then over to 42 for 25mins.
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