RNA extraction from blood is a bit difficult because RBCs are nuclei less therefore extracted RNA will be only from WBCs (PBMCs). It is always better to separate PBMCs first from whole blood and then extract the RNA from PBMC only which reduces the contamination like EDTA or heparin in your vaccutainer, which may hamper downstream processes like cDNA synthesis.
For future RNA extraction you will preserve your PBMCs in RNAlater solution (in -80) rather whole blood.
Never add trizol to whole blood. First isolate WBCs from the blood as recommended by Abhijit Maji..then continue with adding trizol to your cell pellet.
if you are planning to perform RNA isolation on finger pricked blood, I would recommend you to never do that as finger tips contains the enzyme RNase which will degrade the RNA.
I'm learning a lot here. Thank you.
I hope you don't mind me asking one more question:
If Trizol was added to whole blood and frozen, which I just learned I shouldn't, how can I proceed with RNA extraction? Is there any way of salvaging the sample? Can I perform the extraction and then do a cleanup step afterwards?
first of all collect your blood sample in EDTA vaccutainers containing RNA later.. you can add some RNA later in the tubes yourself using a syringe. RNA later has RNA preserving properties. Store the sample in -80C. Then you can proceed with RNA isolation whenever you wish to..
If RNA later is not available then collect your blood sample in EDTA vaccutainer. you can store the sample for 2-3 days at 4C but try to process the sample asap. Do not store the non-RNA later containing tubes in -80C as it can lead to cell lysis due to freezing and thawing.
you can find the protocol online..i am attaching one here..
https://www.lerner.ccf.org/gmi/gmb/documents/RNA%20Isolation.pdf
Is there anyone who is trying to isolate RNA from blood serum as well?
Blood serum is is the component that is neither a blood cell (serum does not contain white or red blood cells).Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances (e.g., drugs and microorganisms).Therefor there is no DNA/RNA in the serum to be extra
Actually some people in their DNA/RNA isolation protocol refer blood as serum which is a wrong practice. If you want to isolate RNA from blood, first isolate the WBCs from the whole blood and then continue with trizol treatment.
Dears
I have question
I want to isolate RNA from human blood. What is the protocol for that without using RNase inhbitor. Should I centrifuge the samples before addin triazol.
Regards
separate the WBCs from whole blood using ficoll and centrifuge it at 2500rpm for 10 minutes. then take WBCs out in eppendorf and add trizol.
GOOD. separate WBCs from whole blood and centrifuge and take WBCs carefully and add trizol.
To extract of RNA from whole blood better to use EDTA tube, then lyse the whole blood to seprate the WBC. After lysis, add TRIzol to the islated WBC. Then finally you can either store under -80 C ro extract directly follwing trizole extraction method.
Note that you have to use also RNAse A and wash before adding TRIzol to exclude other free RNA. But, using the serum we can extract cirulating miRNA.
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