During RNA isolation DEPC and TRIzol reagent is used. How do they work and how is DNA separted from RNA?
Dear Utpol,
DEPC is a strong inhibitor of RNase and it protects the RNA integrity during isolation. whereas TRIzol is monophasic solution of phenol and guanidine isothiocyanate and it is designed to isolate separate fractions of RNA, DNA, and proteins from cell and tissue samples
Vivek Singh is right.
But the reason why RNAs get into the aqueous phase is because the phenol in TRIZOL is buffered with acid. If it was a neutral phenol (buffered at pH7-8), RNA would end up in the organic phase.
I disagree with you Vincent, though I didn't vote you down. I think it is bad education to hide and not bring something to all, so as we can all learn. Anyway, I think at acidic pH only RNA is soluble. So, most of the DNA is precipitated. At neutral pH you will have both DNA and RNA in the aqueous phase. Of course I can be mistaken. It has been quite a time I didn’t perform such an RNA extraction.
Abdelhalim is correct. Nucleic acid (mRNA, DNA, rRNA etc) will be in the aqueous phase at neutral pH. When Acidic, as is the case with TRIZOL, the double stranded nuclear DNA precipitates and ends up mostly in the interface. The phenol is an organic solvent and the guanidine isothiocyanate is a chaotropic agent that denatures proteins. When all of these are present the RNA is safe from degradation. However, if they are still all present you can 't do any enzymic steps as the enzyme will also be damaged.
This leads to the final RNA being resuspended in water which has been treated with DEPC. The DEPC is used to prepare the water by destroying the RNAses. Most, if not all, then autoclave the water to get rid of the DEPC (it gets converted to ethanol and CO2). To DEPC treated water used to resuspend the RNA, does not normally have any DEPC left by the time it is put into contact with the RNA. DEPC convalently modifies histidine so it has to be removed before any solution comes into contact with proteins you want to function e.g. reverse transcriptase.
A last point of interest, don't use DEPC with Tris buffers.
Good luck
WHAT ARE THE FUNCTIONS OF sodium citrate, N-laurosylsarcosine , M 2-mercaptoethanol IN TRIZOL REAGENT FOR RNA ISOLATION ?
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