I am studying a novel transcript in yeast and would like to know if my RNA has a 5'7mG cap. I have tried treating RNA with a commercial cap-dependent RNase (Terminator exonuclease/Xrn1) but it showed sporadic and non-specific activity resulting in degradation of several non-substrate transcripts. I am looking for an alternative method that is compatible with northern blotting or RT PCR like techniques. Thanks in advance.
Pietro's suggestion is very good. Alternatively, you can devise a strategy based on selective dephosphorylation which avoids RNases:
1. dephosphorylate your total RNA sample (e.g. CIP), so that anything that is 5' capped is protected
2. decap the sample, so the only RNA species with a 5' phosphate are the ones that were capped in the first place.
3. Assay whether your RNA of interest is 5'P, e.g. ligate a linker and RT-PCR.
Good luck!
I'm not too familiar with yeast and the tools available, but I would suggest a RIP (RNA IP) using an anti-eIF4E antibody (there are several, from Sigma, Santa Cruz, etc, but I don't know if they recognize the yeast protein, you might know better where to look!), followed by RNA extraction and RT(q)PCR of your specific gene. Comparing with the input and using a negative control (an mRNA that is known not to have a 7-methyl cap) and a positive control (a gene well known to have the cap) you should be able to solve the problem.
A similar approach was used for different purposes in this paper:
http://www.nature.com/nsmb/journal/v20/n6/full/nsmb.2576.html
Good luck
Pietro's suggestion is very good. Alternatively, you can devise a strategy based on selective dephosphorylation which avoids RNases:
1. dephosphorylate your total RNA sample (e.g. CIP), so that anything that is 5' capped is protected
2. decap the sample, so the only RNA species with a 5' phosphate are the ones that were capped in the first place.
3. Assay whether your RNA of interest is 5'P, e.g. ligate a linker and RT-PCR.
Good luck!
The Epicentre 5' and 3' RACE kit is probably what you want. It lets you oligo-cap only 5'm7Gppp mRNAs and distinquish from the monophosphorylated species. I used this kit last year and it was very straightforward. You can omit the tagging of the poly(A) if you want and just design a gene-specific reverse primer to use in conjunction with the oligo-capped primer for PCR. http://www.epibio.com/applications/rna-sequencing/transcriptomics-(tn5)-mutagenesis/exactstart-eukaryotic-mrna-5---3--race-kit?details
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