I am seeking to isolate miRNA from serum using Qiagen serum/plasma advanced kit - after running the smRNA bioanalyzer QC on the RNA only 60mer and higher are represented with no 20mer. Why is this happening?
I am having the same issue with miRNA from whole murine blood using Qiagen animal blood kit, the DNAseI digestion done on the whole blood samples and not on serum.
Why would I lose everything below 60mer? I am not losing all smRNA. I have tRNA and rRNA. Attached are the bioanalyzer data
serum (1-11) and blood (Samples 1-10 only ) are attached. any input is greatly appreciated.
all steps in the kits for total RNA including miRNA were strictly followed, including standard protocol for RNAwork
Thank you!
Did you do these BioAnalyzer runs yourself? There's something a bit odd about the blood one. The small RNA BioAnalyzer marker is a 4-mer, and the instrument has listed the assay class as small RNA for both the blood and serum runs, but in the blood run it's showing the marker peak (green lines) detected at 35nt. I don't know how this can happen when the assay class is correctly selected, but I would consider repeating this run if you have enough sample.
I've extracted a lot of small RNA from mouse brain and cultured cells, and in my experience there's often no clear band/smear at the ~20-30nt size range. Even when I can see nothing at all on the BioAnalyzer, typically a strong miRNA band is then detected from subsequent library preparation. I do think it's odd to see inconsistency in your samples (in the serum samples there's a clear band at ~20-40 in some samples but not others) but if you can think of an explanation for that which wouldn't affect sample validity, I think going ahead with a library prep and seeing what comes out would be a reasonable thing to do.
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