Hi, I have been doing RNA isolation from human EDTA plasma using TRIzol LS and I am having some issues with the RNA quantification... I have measured RNA amount (by using 1µL of sample) with Nanodrop 2000, Qubit 4 Fluorometer and, Agilent RNA 6000 Pico.
I didn't obtain same results as you can see...
- I didn't mention Qubit in my table because RNA is to low to be quantify (
Nanodrop instruments typically don't have good accuracy for samples with low concentration. I would trust the other instrument more, though Agilent's pamphlet for this suggests the range is 200-5000pg/uL.
You may want to consider using a common "house-keeping" RNA that you don't expect to change and quantifying that by a PCR-based method - it should at least roughly tell you relative levels of RNA that you have. You may also consider using more plasma to start with, and using a combination of Trizol-LS and column-based purification kits (such as Qiagen's miRNA or RNeasy kit).
Good luck!
Thank you all for your anwers, I will compare Trizol LS to column-based purification kits !
Murtaza S Nagree, few months ago I have tried to do RT-qPCR using a common house-keeping RNA, but I didn't have relevant results. I suppose that was due to the fact that circulating RNA are mostly small or fragments. Maybe I could use a known cirulating miR.
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