which kit(s) to use for peripheral blood mRNA/miRNA

[bjloftus]

Hi,

Just wondering if there is one kit that would cover mRNA/miRNA from peripheral blood samples and if so what would be a good depth of coverage..or if the sample needs to be split into two seperate pipelines

Thanks

[adam.geber]

I've done entirely satisfactory RNA extractions with both the Ambion RiboPure blood kit and Qiagen's miRNeasy kits. Just make sure you're doing everything possible to retain your small RNAs! I believe Qiagen has recommendations available on how to increase the binding affinity of RNAs <200nt.

As to whether your individual samples will be useful for both mRNA and miRNA, you'll certainly have to do your library prep separately because of the size selection step for small RNAs. I've wondered if you can combine these enrichment procedures given that the miRNA and mRNA profiles shouldn't overlap in theory -- perhaps someone else can weigh in on this? I typically split my total RNA samples according to the input mass requirements for polyA or miRNA enrichment, concentrating the miRNA portion as necessary to meet volumetric requirements for e.g. the TruSeq Small RNA kit.

[kerplunk412]

I agree that you definitely need to split your library preps for miRNA and mRNA. There is no kit that can do both simultaneously, for now. Most small RNA library prep kits work fine from total RNA, so you probably don't need to split your RNA prep as long as you use a method that isolates total RNA including small RNA (as adam.gerber noted this may require a modification to the default Qiagen protocol, just adding more ethanol at the binding step).

As for read depth, a million per library is usually enough for small RNA, and 10-30 million per library is typical for mRNA, depending on your goals.

[bjloftus]

Great informative answers and I appreciate the time taken!