Hello
I have to measure blood RNA and I would like to ask for advice on whether it is better to use whole blood or eliminate red blood cells. What is the best strategy? How much do red blood cells influence total blood RNA?
Thank you very much for the help!!
Hello,
Phenol:chloroform extraction of RNA from blood should be fine (even in presence of RBCs)
https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/purify-rna-directly-from-whole-blood.html
If you do not want to buy the kit, here is the recipe of RNAlater‐like buffer solution: For 1.5 liters: 935 ml of autoclaved, MilliQ water 700 g Ammonium sulfate Stir until dissolved Add 25 ml of 1 M Sodium Citrate And 40 ml of 0.5 M EDTA Adjust to pH 5.2 using concentrated H2SO4 (about 20 drops= 1 ml) Store at RT
Hope it works
In my opinion, The RBCs do not hamper total RNA extraction anyway. But if you wish to extract RNA only from WBCs, you can separate them by fractionation. For this, centrifuge the whole blood at 3000-3400 rpm for 10-15 mins and remove the WBCs (a ring like layer between lower RBCs and upper plasma layer). Mix the WBCs with RNAlater to stop degrading RNAs for upto 5 days or proceed immediately (if you proceed immediately, RNAlater is not necessary).
Since your objective is epigenetic study, any method that extract total RNA would be fine. Total RNA because you may also need small RNAs (such as micro-RNAs), which are the important epigenetic regulators of gene expression. So, it is better to have them in your samples in case you wish them to study. Since Phenol-based isolation procedures (such as TRIzol reagent :Chloroform extraction) extract RNAs from 10 to 200 BPs, you can use it. I generally get higher concentration of RNAs using TRIzol extraction, so I prefer it. If you use the silica matrix column or GFF procedure (i.e. Qiagen RNEasy columns), it will significantly deplete small RNAs, hence I do not use them for epigenetic studies.
Hope this will help. Goodluck.
Better the whole blood cell according to my view as scientist after i studied genetics
Thanks Savita, Prabin and Meshack!! I will try your suggestions!!!
Mariela
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