Hi all,
I am doing some RIP-qPCR or CLIP-qPCR to verify some binding sites of a specific RNA binding protein. Since native RIP does not include crosslinking or RNA trimming, the whole transcripts bound by the RBP will be pulled down. But I want to precisely locate the binding sites within 3' UTR but at different regions. So, I include crosslinking and RNase T1 digestion. I did some pilot experiments to find optimized RNase T1 concentration to have RNA fragment sizes around 400-600 nt. And usually, my qPCR amplicon sizes are around 100-150 bp. I designed some across the predicted binding sites. But, the results are not ideal. Please share your experience at primer designing, or optimization of the whole RIP procedures.
Many many thanks and happy holiday!
Lu
Hey Lu,
I used a native RIP protocol to pulldown a lncRNA that was attached to an RBP. I detected the RNA in the pulldown samples by qPCR. For this, I used the "normal" qPCR primers that were used in conventional qPCR on total cell RNA extractions. This worked well in my hands Article The lncRNA CASC9 and RNA binding protein HNRNPL form a compl...
Retrospectively, I would have preferred to use RNA sequencing of the RIP samples. By this, you would avoid the whole primer designing issue. In addition, you could identify additional RNAs in an unbiased way that were not considered to be bound by the RBP. The distribution of the reads will probably show you the exact binding sequence Article RIP-Seq data analysis to determine RNA-protein associations
Best,
Marcel
Thank you Marcel, that is helpful.
Actually, I want to specifically detect where my RBP binds in a long 3' UTR region. for example, within a 2kb range, I would like to know some specific regions, maybe around 100-200 bp where I can design primers to check in qPCR.
That is why I didn't use traditional native RIP, I included crosslinking and RNase T1 partial digestion to narrow down the binding regions.
Thanks anyway! And good luck on your research.
Lu
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