I am trying to RACE clone a transcript using Clonetech Smarter RACE kit which is for RACE clone of mRNAs. I successfully identified the 5' end of this transcript but failed for the 3' end. After checking some sequencing data I realized that it might be a transcript without polyA tail. Any ideas about how to RACE clone the 3' end of a transcript without polyA tail? Any commercial kit available?
Thanks in advance
One possible approach is to ligate a linker and use it to prime a reverse transcription and PCR. Not sure about kits, but it is sufficiently similar to making small RNA libraries that I would have a look there. You can even use a partly stem-loop oligo that would ligate AND prime. Chemistry-wise, if you are not using a kit, it is best to ligate RNA to RNA, so use a DNA oligo with a few (2-3) RNA bases at the 5' end.
Let me know if you need further clarification or if this is sufficient to set you in the right direction :-)
Good luck!
We are using a standard protocol without Kit for 5' und 3' RACE, if you are interested I can provide the protocol for you.
Just let me know.
@Anna, thank you very much for your suggestion! I noticed some papers used the miRNA cloning method for race cloning polyA lacking transcripts. So it should work but I still need to think about the details.
@Ulrike, sure I'd like to have a look at your protocol to see whether it's applicable for polyA lacking transcripts. It will be great if you can share the protocol with me!
@Kejun, thanks for you answer but I need to know more information about how the primers were designed
you can try degenerated primers. To design them you only need the nucleotide sequence of your gene in other species (the closer the better), then align them to find the conserved regions and design primers in those regions.
ask for details if you wish.
good luck!
HI Xiaona,
You can also use a two step approach based on existing established methods
1) Capture the polyA tail containing transcript from the total RNA pool by first binding them to the biotinylated oligodT, subsequently to the tubes coated with streptavidin (eg mRNA capture kit provided by Roche). This is generally used for enricing the mRNA from the total pool by discarding the non-bound transcripts.
2) For your requirements you need to collect the non-bound transcripts, precipiated them and
3) Add poly A tail by PolyA polymerase enzyme or terminal transferase.
4) Use standard OligodT based reverse transcription for 3'RACE protocol..
it works
bye
Hi Xiaona,
did you have a look at the protocol? Was it helpful? If you have more question feel free to ask.
Ulrike
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