Does anyone have experience assessing RNA quality after RNA-immunoprecipitation? My downstream goal is RNA-seq (RIP-seq), and I want to check the quality of the RNA before proceeding to library preparation.
I've run my eluted, DNAse treated, and purified (Zymo Clean & Concentrator) RNA on a Agilent Bioanalyzer RNA Pico chip.
To MUCH dismay, my RIN was very low ( ~2) with very low concentrations.
Before trashing the very difficult to obtain samples, I just want to know if the Bioanalyzer is an accurate indicator of RNA quality when that RNA is isolated from an immunoprecpitation experiment. Any input would be great.
Hi Samantha, Yes Bioanalyzer is so far the near best way to analyze the quality of RNA. Using nano drop will only give information about the quantity but it will not give indication about the fragmentation in RNA. For your RNA-seq you would probably need intact enough RNA (lets say of RIN around 7). You can give a try with RT-PCR . This is only we can do with your RNA. RNA is always hard to deal with if you are not very used to do RNA extractions. There are a lot to consider like speed of process and de-contamination with RNAse free. Loading of chip and calibration could also sometimes creates problem in bioanalyzer.
If your aim was to amplify your RNA, RIN of 2-3 is ok for that.
Hi, Samantha.I'm working with plants and use antibody-free system in my experiment, but this article helped me. Authors determined RIP samples (without cross-linking) quality on a Bioanalyser.
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