Hello,
does anyone have experience with the QIAamp Viral RNA Mini Kit? It uses poly(A) carrier RNA to enhance gain of RNA extracts. I use the kit to extract viral RNA from bees, and wondering whether the carrier RNA will be present in the extracted RNA or if it will be washed through the membran, i.e. are the carrier poly (A) chains long enough to be hold on the membrane and present in the isolate or shorter than the min. 200bp and washed out? The question is whether the carrier RNA effects NGS down stream analysis like RNA-Seq.
Thanks
Benjamin
Hi Ben, what do you use for the extraction? Whole bees?
According to the manufacturer: "The QIAamp Viral RNA Mini Kit simplifies purification of viral RNA from cell-free body fluids with fast spin-column or vacuum procedures."
Have you tried with hemolymph?
Hey Alex, nice to hear from you. We use whole bees, grind them in liquid N2, elute the powder in phosphate buffered saline and centrifuge debris down to achieve a cell free supernatant for extraction. Because the RNA yield is low in that case QIAamp uses a poly(a) carrier RNA for better precipitation. I basically want to know how this carrier effects RNA-seq down stream analysis.
Thanks Mathew for your answer. If the carrier RNA is present in the extraction, I guess it would waste some sequencing capacity? The other thing is, how much of it ends up in the transcriptome (we do assembly) also depends on the kind of first strand cDNA priming. We use random primers, so probably some carrier RNA would end up in the library, but sould be easily recognizable as poly(a) sequences, I guess?
kind regards
Here is the answer from Qiagen technical service:
"Our carrier RNA is a poly(A) RNA, produced in a biotechnological production process, in the size range of 100 to 10,000 base pairs and therefore it will be present in your final RNA eluate. The use of carrier RNA is not recommended during RNA purification for RNA-seq since it may interfere with your downstream application."
Can we used carrier RNA in any kind of lysis buffers of varying pH or they work at specific pH only?
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