I have been using miRNA Isolation mini kit (qiagen 217004) for isolating RNA from PBMCs stored in RNA later at -80 C. The cell no. was 2 million cells. The RNA yields obtained are around 30-40 ng/ul and the purity around 1.4-1.5. I have tried doing some necessary steps in order to increase the RNA yield such as complete removal of RNA later, complete homogenization of the PBMC pellet, re-elution of the RNA using elution buffer. What could be the reason behind the low RNA yield and what steps can I take in order to rectify it?
It may be too late for this response to be helpful... However, for what it's worth, our lab has had better luck (in terms of yield, purity, and quality of RNA extraction) storing cells/lysate in the RLT Lysis buffer that is provided in most (all?) of the Qiagen RNA prep kits rather than in RNA Later. This prep is then stored at -80C until the additional RNA isolation process can be completed.
I have attached two white papers (Miltenyi Biotec and Qiagen) that I thought might also help (in terms of expected yields from PBMCs).
I hope some or all of this info might be helpful! Best of luck to you!
Melissa
Note: Prithy Martis any other tips you might be able to provide?
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