5'RACE of RNA Pol3 transcripts? - (Sep/13/2012 )
I found an interesting Antisense transcript running opposite my gene of interest. So i performed 5'RACE to attempt to map the TSS of this transcript, but after two months, and a variety of different primers i am unable to get a band that corresponds to my transcript. i am getting bands, but upon sequencing, they are not the sequence i was looking for.
There is a U6 snRNA just upstream of this transcript, which is transcribed by RNA polymerase3, so maybe my transcript is also a Pol3 transcript.
So i was wondering is 5'RACE possible with RNA pol3 transcripts? and if not is this the reason that my 5'RACE has failed?
Thanks for your insight.
How do you run your 5'RACE? what is your protocol step by step?
I isolate RNA from tissue, DNase, and then using the Applied biosystems High capacity cDNA synthesis kit, synthesise first strand cDNA, using a gene specific primer for the antisense transcript. i then add a polyA tail to the 5' end of the transcript using dATPs and Terminal Transferase. I clean up the cDNA, and then perform a nested PCR, using a anchor primer with a PolyT tail, and a internal gene specific primer. then a second round of PCR using a third internal nested gene specific primer and a primer that binds to the anchor primer.
I think that this antisense transcript may be endogenously transcribed by RNA pol3, and my question is,
would this 5'RACE protocol work on a RNA pol3 transcript, when it was original designed to amplify the 5' end of a RNA pol2 transcribed transcript.
thanks so much for your help
By cleaning up cdna you lose a lot of it. have you tried running pcr without cleaning up your cdna?also,have you checked the tm difference of your primers?