I want to define both 5' and 3' ends of a lncRNA using GeneRacer Kit (Invitrogen). I don't know if my transcript has a poli(A) tail and the manual recommend using an oligo(dT) primer to reverse transcribe the mRNA if I want the sequence information for the 3' end...I wonder if the GeneRacer RNA oligo that we ligate to the deccaped mRNA is enough to the oligo(dT) primer to anneal and synthesise my cDNA (in case it is not polyadenilated). Can anyone help me?
Maybe you can try random primers (Random Hexamers - Invitrogen) for reverse transcription instead of oligo(dT)?
In some kits for RLM-RACE (like the Ambion catalog number AM1700) random primers are used instead of olido(dT).
Good luck!
I was going to, but then I read the manual again and they say "If you do not need information for the 3' end, you may use the Random Primers provided in the kit or a GSP to syntesize cDNA." Since I need the 3' end, I got confused about that information...
But thanks for your help!
The GeneRacer RNA oligo is basically just a 5' RNA adaptor and will be ligated to the 5' end of your RNA to provide a known sequence at the 5' end and thus allow you to determine the exact 5' end of your transcript.
To determine the 3' end of the RNA, you can use an oligo(dT) primer if the RNA has a polyA tail. If you are not sure, you can simply add a polyA tail yourself using poly(A) polymerase (NEB #M0276S). I would also use an anchored oligo(dT) primer, e.g.: 5'-(dT)30-VN-3', with V being any nucleotide but T and N is any nucleotide.
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