Dear all,
Few years ago, I used miRCURY Biofluids from Exiqon for extracting RNA from plasma for detecting miRNA by qPCR. However, this product is currently discontinued since Exiqon is now integrated with Qiagen. So from your latest experience, what is the best method to extract RNA (including miRNA) from plasma if it is needed for NGS RNA sequencing? do you use a kit for extracting only miRNA such as NucleoSpin® miRNA Plasma (MACHEREY-NAGEL)? or do you use a kit for extracting the total RNA such as miRNeasy Serum/Plasma Advanced Kit (QIAGEN)?
I am wondering if you use the total RNA for NGS sequencing, high proportion is mRNA, have you any idea/experience how that could affect to the sequencing of the small RNAs?
Thank you,
Best,
Salam
Hello Salam,
Normally the miRNeasy works fine for qPCR or NGS analysis. If you contact Qiagen I think that you may be able to find some remaining stock of MiRCURY (I did so). Now if you want to have all lengths of RNAs, you can extract the RNAs by using the standard TriZol protocol. It works fine, you will have a lower yield of miRNAs but this allows you to include all kinds.
Regards,
Nikiforos
Hello Salam,
Normally the miRNeasy works fine for qPCR or NGS analysis. If you contact Qiagen I think that you may be able to find some remaining stock of MiRCURY (I did so). Now if you want to have all lengths of RNAs, you can extract the RNAs by using the standard TriZol protocol. It works fine, you will have a lower yield of miRNAs but this allows you to include all kinds.
Regards,
Nikiforos
Dear Salam,
Regarding the miRNAs from plasma samples we have very good experience with Plasma/Serum miRNeasy kit and subsequent QIAseq miRNA Library kit. We have tried CleanTaq Small RNA Library Preparation Kit (TriLink) with the same extraction kit in the past but the miRNA results were very biased mainly due to artificial over-amplification of miR-486-5p (sometimes 99% of all miRNA reads!) therefore I don't recommend this one (TriLink) for plasma/serum samples if you go after miRNAs.
Dear Jan,
Thank you very much for the useful information!
Did you also try the new kit miRNeasy Serum/Plasma Advanced Kit which is phenol-free isolation kit? and do you have experience in detecting other ncRNAs with miRNeasy kit? I am wondering if we could also detect other circulating RNAs in plasma because in the description of the kit it says that is for the extraction of total RNA including miRNA.
Thank you
Best,
Salam
Dear Nikiforos,
Thank you very much for your recommendations! I thought we could also use miRNeasy kit for detecting other RNAs since in the specifications of the kit it says is for total RNA extraction including miRNAs. Does TriZol-LS extraction of RNA yield with good results in plasma? I used TriZol for the extraction of RNA from cells or tissues and worked good but I am wondering if it works fine with biofluids?
Best,
Salam
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