11 November 2019 2 4K Report

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

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