3 replies to this topic

[jaywilla]

Hi Guys,

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.

[Curtis]

How do you run your 5'RACE? what is your protocol step by step?

[jaywilla]

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

Edited by jaywilla, 14 September 2012 - 01:24 AM.

[Curtis]

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?