Hi everyone! I work with Mesenchymal Stem Cells-derived exosomes and I am interested in doing next generation sequencing of exosome-derived small RNAs. After isolation, I obtain a very small amount of RNA. How can I corroborate the integrity before I send it to sequencing? Checking in agarose gel is not an option since there is not ribosomal RNA in the sample.
Thank you in advance!
Hi, exosome-derived small RNAs are different from cell total RNAs. So the RNA integrity number (RIN) can not evaluate the RNA quality. In my opinion, you could isolate exosomal RNA together with several cell total RNA samples as a control, which can tell you that no mistakes and problems appear during the purification.
Do you have access to a BioAnalyzer? The small RNA chip type works from tiny amounts of sample and shows good resolution in the 20-200nt size range. If you have a positive control (exosome samples that are known to be of good quality or work well for your application) you could compare your samples to that. Otherwise, you could check that they all look the same, which would tell you that your isolation method is consistent. If there are any specific small RNAs that you know definitely should or definitely should not be present in your samples, you could check for those using qPCR as an additional control :)
Exosomes contain extremely small amount of RNA, this is normal. For qPCR one can do "blind input" - ie you can go ahead with isolated RNA without assessing its yield or quality. NGS is different as more RNA is required and the procedure is expensive. So ideally you have to process more sample- to get a decent amount of exosomes and RNA cargo, enough for Nanodrop or Qubit analysis, and Bioanalyzer. eg for serum/plasma- use 4 ml or more.
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