Does anybody have experience with isolation of RNA from a small sample? I am trying to magnetically separate CD4 cells from whole blood and thereafter isolate total RNA from that small cell population. I have found Norgen RNA/DNA purification kit which is characterized for difficult samples, such as small amount of cells, has anybody tried to use this kit and would you say it is good for purpose I am looking for?
Hi Maja, how to purify your RNA will depend on what you need to do with the RNA afterwards and how many cells you have.
If you need pure RNA, then the Qiagen RNEasy Micro Kit, or similar, will yield pure RNA from small samples ranging from dozens to 500k CD4 cells.
Are you going to make cDNA and do RT-PCR? If so, then I would recommend the Cells to Ct Kit by Ambion (https://www.thermofisher.com/au/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/total-rna-extraction/cells-to-ct-kits.html). This kit avoids the need to separately purify your RNA and generates cDNA for you to use in RTPCR. You could consider a preamplification PCR reaction before RTPCR if using very low amounts of cDNA (e.g. Taqman PreAmp Master Mix).
Best of luck
Hi Maja,
I haven't tried that many kits, but my experience is that the best method to isolate RNA from small samples is the manual extraction (no kit, old fashion phenol-chloroform protocol). It provides the best yield.
All the best,
Catherine
Thank you for your help,
Peter, I have considered preamplification step before RT PCR and I think I will do it. Ct Kit by Ambion is something I haven't found before, but it seems promising, I will definitely think about using it.
Catherine, thanks for sharing your experience. For now I will hold on commercial kits, but if my results will still be bad, I will try the organic old fashion way ;)
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