Dear all,
My lab hypothesized a new potential RNA-binding role for a particular protein. We'd like to see whether it binds or not to RNA. Since we don't know if it binds to mRNA or any kind of RNAs, we preferred to use an unbiased approach.
Is there any way to amplify those unknown RNA sequences for sequencing?
Thank you for your time!
Other than by generating a library of the amplified products I don't see how. Without primer or template specificity your sequencing will be a mess. This comes with the caveat that I'm an old school sequencer and those new fangled NGS chaps might have a solution that I don't even know about.
You can use generic or specific primers defending on your target gene.
That's the problem here. We don't know what kind of RNA binds to our protein (can be miRNAs, siRNAs, mRNAs, snRNAs, etc...). Therefore, we can't use specific primers since we don't know the nature of the RNA (neither the sequence). We don't want to use random primers as well since it will introduce a lot of biases if only small RNAs are binding to our protein candidate.
You can do RT PCR to get your DNA and send for sequencing from the sequence obtained you can design the primer of interest.
RNA obtained following RNA-IP is normally too low to even be detected by spectrophotometry. Also, if miRNAs are binding my protein candidate, it is quite hard, to not say impossible, to do an RT-PCR without knowing the sequence at first (Varkonyi-Gasic et al. 2007).
My RNA will be from a Eukaryotic nature. Once again, since they might be small RNAs, those not being genes, I can't design specific primers for my gene of interest.
For any potential mRNA you could use poly T to prime from the poly A tail.
Dear Artur,
My protein was structurally predicted to bind RNA. I have the RNA at hand (or not since the concentration is too low to observe it). Normally, following RNA-IP, the concentration of RNA is too low to perform NGS library and sequence. We need to amplify at first and then do an NGS library.
Could you ligate your RNA-IP sample to a synthesised RNA from which you could prime RT and THEN generate a cDNA library? I'm guessing it'll be fairly messy but in the absense of anything else...........
For whom wondered about the answer to that question, I just got a good answer from a guy at our genome center. This kit can be used to amplify every kind of RNA with an amount as low as 50 pg. The RNA obtained after this amplification is supposed to be crystal clear.
https://www.diagenode.com/p/CATS-RNA-seq-Kit
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