need to find the real peaks for mi RNA and pi RNA before size selection procedure , i am not feeling confident enough for proper size selection step by amppure beads, imporantly dont want to go for gel based purification. please share your experience for such experiments, so that i can follow them to get proper enrichment of MiRNAs and piRNAs?
Hi Cash Kumar,
I can only help in determining the miRNA peak. Based on my experience working with miRNA samples for NGS projects, the miRNA peak lies before 200 nt (equivalent to the small RNAs peak in mirna-2.jpg). Hope it helps you.
hi Boon , thanks for reply , i am facing issues of adaptor dimer , after bead based size selection. can u identify those too ? u r showing 200 bp peak as mi RNA peak but in generaly it comes at 140 -150bp
Hi Cash Kumar,
If you refer again to mirna-2.jpg I attached above, the peak of small RNAs is below 200nt. I attached Additional file 1 for your perusal to better identify bioanalyser electrophoresis (with ladder) and electropherogram of intact RNA samples, i.e. RIN number at least 7. Only intact RNA samples can be used for NGS library construction and sequencing. All the attachments I showed you are just Bioanalyser assessment on "Total RNA samples containing small RNAs".
Sorry I couldn't help you with the adaptor dimer issue post bead based size selection. I used gel based size selection of small RNA for my work.
" View the gel on a Dark Reader transilluminator to avoid being exposed to UV light. The SRA ladder is in 20 bp steps up to 100 bp. Using a clean scalpel, cut out a band of gel corresponding to the 18-30 nucleotide bands in the marker lane. "
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