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Please Tell whether RNA IP (RIP) from E. coli is it feasible

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#1 Myco Rax

Myco Rax


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Posted 20 April 2011 - 02:01 AM

Dear All,
My protein of interest is an RNA binding protein.
It specifically binds to certain responsive elemnts (RNA secndary structures).
I have to identify which all genes hav these Responsive elements in their UTRs of mRNA.
what i planned to do is,
Take the E. coli cells with protein constrct and induce expression using D-Arabinose. then Crosslink the RNA-protein interactions with 4% HCHO/Methanol (10min) and neutralize it with 0.25M glycine (15min).
Then lyse the cells by sonication (20S pulse on and 1min Pulse off).
then perform Immunoprecipitate my protein under RNase free conditions.
then reverse the crosslinks by heating at 70 degree C.
then isolate the RNA by trizol method.
I have to then prepare cDNA. As i want all the RNA bound to the protein, i tried using Oligo dT and also random primers separately.
Then ligate the single strand cDNA with a modified (3' modified) oligo with T4 RNA ligase.
then tried to amplify the cDNA with the primer complementary to oligo and oligo dT/random primer.
but i cud see no amplification in any case.
can anyone please suggest whether the method is feasible with E.coli.
And please suggest any alternative other than microarray (cannot afford the tech).
Thanks in advance.

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