I am isolating total RNA with miRNeasy micro (PBMCs) miRNeasy serum plasma (serum) and exoRNeasy (serum/exosome) for Next Gen Sequencing for miRNA. I believe my final product should contain mostly miRNA.
What is the best way of determining total RNA in such circumstances? I can't use nanodrop as it is not good at low concentrations I am expecting and miScript PCR assay doesn't fit my protocol. I wanted to use the Quant-it miRNA but the sequencing people are asking for total RNA.
Thanks
Hi Sahin,
I am confused on the question?
Total RNA means all the RNA in the sample, which including miRNA.
Do you mean to measure all other RNA except the miRNA?
no, I mean measure total RNA.
Quant-it RNA and Quant-it Ribogreen are not sensitive to small RNA species, and Quant-it miRNA is not sensitive to large RNA species. in both cases I am losing some of my population.
Hi Basak
For RNA in biofluids, I am afraid that you cannot use conventional systems like Nanodrop for quantification. The best choice will be to use capillary electrophoresis devices like Bioanalyzer.
If you are planning to do small RNAseq, you should extract total RNA by conventional methods as you described, and quantify it by Nanodrop or Bioanalyzer. Afterwards the people from the NGS service will separate the small RNA fraction (
we recently (in our lab) started using the fragment analyser to measure microRNAs in biofluid samples, it is much easier and more convenient then bioanalyser and it can give you the total RNA conc. in the sample as well as how much of this RNA is microRNAs.
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