I'm extracting RNA from PBMCs, and do not know what constitutes a normal yield.
What is normal for your lab, what method do you use (TRIzol, RNeasy, etc.), and how many input cells do you use? Also, what is your downstream application (e.g., qPCR, microarray, RNAseq), and does that inform your choice of extraction method?
Many thanks!
I've used both column purification and trizol for RNA seq. Both work well, though I've read that some micro RNA's are lost through the column method. If cost is an issue I'd recommend using trizol (for liquid cultures) Just make sure to treat your RNA with DNAse before reverse transcription. It's usually a good idea to check the purity/concentration (nanodrop) and the RIN (bioanalyzer) before sending away for sequencing.
We routinely do RNA extraction for qPCR, from PBMCs using spin columns. PBMCs from 1.5 to 2 ml blood gives 150- 180 ng/ul RNA.
Dave, are you extracting from PBMCs? If so, what is your yield, and how many input cells are you using?
Neha, are your blood samples from normal controls? We observe pretty large variation in the PBMC yield from whole blood within our patient cohort.
Neha, how you purify the PBMCs from the whole blood for isolation RNA?
Amanda, i do not perform any purification step... Buffy coat obtained after density gradient centrifugation is washed once with 1x PBS and RNA is isolated after desired treatment.
Hi Carl. It just depends on your input. Here is a good protocol for isolation methods and storage, specific to PBMCs.
http://www.gene-quantification.com/eikmans-et-al-rna-quality-blood-2013.pdf
and the manual from Trizol indicating typical yields (60-70ug for 10^7 cells)
https://tools.thermofisher.com/content/sfs/manuals/trizol_ls_reagent.pdf
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