Hello to all,
We are trying to isolate miRNA from CSF (glioblastoma patients) and the yield is really low. We used Qiagen kits, Trizol (Invitrogen) and we added glycogen.
I just would like to ask you if someone was successful with extraction of miRNAs from CSF? Which kit did you use? How big input? What was your yield?
And could I also ask you about the yield of some specific miRNAs, you've tried to quantify? Just if you can tell me, of course.
And is there someone who has experience with Exiqon miRCURY Isolation kit?
Thank you very much for all answers
Hi Alena,
In your case, less may be more. My experience with protein rich biofluids (serum) has been that diluting the biofluid increases yield. We use a reagent called RNAzol BD that improves the RNA quality and quantity significantly. Also you may try to isolate exosomes from CSF (has been done before) and try RNA isolation from there. Would definitely reduce the amount of protein you need to deal with and probably improve RNA quality and yield.
AB
Dear Alena,
mIRNA isolation from CSF is challenging because, as you mentioned, total RNA conc is very low (5-30 ng/µl) and the miRNA portion is only about 0.01%. I tried various kits such as miRVANA Paris, miRNeasy, miRCURY, TRizol and Norgen. From my experience, the miRNeasy kit gives you the best RNA yield (200 µl input) - which is also consistent with literature in this area (e.g. Li et al, paper is for serum but it also applies for CSF). You can modify the protocol to increase yield by e.g. use phase-lock tubes, carrier RNA (only minor effect), lower the elution volume, heat the elution buffer or water before elution. Depening on your downstream quantification method, you will be able to reliably detect about 100 miRNAs in CSF (RT-qPCR, LNA Exiqon, w/o pre-amplification), that show reasonable expression levels above LOD. You will surely detect more, but there is no benefit to detect miRNAs which you only see in let's say 50% of your patient samples making your data less reproducible.
JD
If you are using RT-qPCR for miRNA quantification, then try total RNA for cDNA preparation using miRNA specific looped primers (Life Technologies) rather than miRNA, because miRNA enrichment causes significant loss of miRNA. Using looped primer and further TaqMan chemistry will minimize false positives.
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