I did an RNA-IP out of cells and my RNA yield for the inputs was great but the yield was poor for the IP samples. Is there a way to increase the yield of RNA? (other than increasing the starting material; I used a confluent 15 cm tissue culture dish per sample)
How long did you incubate the lysate with primary antibody? Yield can sometimes be increased by incubating for longer, up to overnight at 4C (with RNase and protease inhibitors added). You could possibly also reduce the number or stringency of wash steps, although this can increase the amount of nonspecific binding. Usually it's better to have less RNA that is higher in quality and specificity - if your aim is sequencing, then you only need a few hundred picograms to get a good quality library.
thank you for your response. The purpose of the experiment is to perform an RIP then RT-qPCR in order to validate previous RIP-Seq results. I did the previous experiments at my collaborators’ lab and this us the first time it is done in our lab and my first solo attempt as well. For the IP step, I incubated the lysates (precleared in agarose beads & diluted with lysis buffer with RNase inhibitor & PIs added to the lysis buffer) with FLAG-tagged magnetic beads for 2 hours at 4
degrees with rotation (That’s what we did for the RIP-Seq).
How similar is your current experiment to the previous RIP-seq experiment that worked? If any of the important parameters have changed (cell type and quantity, antibody/beads and quantity, lysate volume, incubation time and temperature) then as a first step I would try changing them back to what they were for the RIP-seq experiment that worked.
If the parameters are constant but the yield is significantly worse, then you might have to start looking for weird causes. Strange things like different brands of plasticware (some adsorb RNA or antibodies or both like crazy!) can cause failures of established procedures that previously worked in another lab.
Another thing to consider is the RNase inhibition. Are you using the same concentration and brand of RNase inhibitor and the same buffer formulation? A lot of RNase inhibitors require the presence of DTT in the buffer. Check the DTT concentration and swap in a different aliquot of RNase inhibitor and DTT because both are sensitive to degradation if they're old or freeze thawed too many times.
Also - are your samples fixed or crosslinked at all? This step is extremely sensitive and often needs re-optimisation to work with different equipment.
Thank you so much for your detailed explanation. I am the only one working on an RBP in my lab and your comments were really helpful.
Everything was essentially the same as the RIP-Seq experiment except for the beads and the magnetic (they weren’t the exact same brand as the ones used by our collaborators).
The RNase was the same one as the RIP-Seq experiment. DTT was added to the lysis buffer and I had prepared everything fresh and using DEPC water.
Thanks again, I appreciate your help! 🙏🏻
And the cells were not crosslinked. We tried CLIP at some point and it didn’t work and that’s why we switched to RIP. we might give the CLIP another try though. Laura Leighton
You're very welcome and I hope you're having better luck! Switching back to the same brand of beads might be helpful if possible.
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