[Q] RT resistance (DEPC, formamide)

David F. Spencer dspencer at is.dal.ca
Mon Mar 23 16:10:50 EST 1998


In article <350dab8d.49272495 at news>, jrt at home.com (John R. Thompson) wrote:

> khlee at MED.CHUNGBUK.AC.KR ("ÃÖ¿õ") wrote:
> 
> ><HTML>
> >Dear Netters:
> >
> ><P>Are the reverse transcriptases (which is usually used in RT-PCR, such
> >as AMV or MMLV RT) resistant against 0.1% DEPC (RNAse inhibitor) and
formamide?
> >
> >
> 
>  If hybridization is the only end use, DEPC is an excellent choice for
> RNAse control.   If enzymes are involved, more careful consideration
> is required.   DEPC is a histidine-modifying reagent.  It kills
> enzymes with HIS residues in the active site (and may react with one
> or two other aa residues to a lessor extent but I can't recall which).
> DEPC is short-lived in solution, breaking down to CO2 and water, and
> is completely eliminated by autoclaving.  It is also an RNA modifying
> reagent and was used as a base-specific cleavage reagent in chemical
> sequencing protocols.  Stating the obvious, base cleavage wouldn't
> help your template.  However, for RNA chemical sequencing, DEPC was
> used at high concentration and elevated temperature (10% and 60C I
> think) to achieve <=1% hits.  So at 0.1% and room temp it may or may

In chemical sequencing of RNA (which is still done, although admittedly in
very few labs any longer) the DEPC is used at a concentration of 1
microlitre per 200 microlitre reaction mix (this is about saturation); the
reaction is done at 90 C for about 5 minutes.  In using DEPC to destroy
RNAses the critical targets are the histidine R groups (imidazole) found at
the active site. The reaction of DEPC with histidine is about 2 orders of
magnitude higher than is the reaction with (also the imidazole ring) in
purines. However the reaction of DEPC with mercaptoethanol is about four
orders of magnitude higher than with purines so mercaptoethanol can be used
to kill DEPC very effectively.  DEPC actually reacts with Tris about 25
times more rapidly than it does with purines so DEPC won't last long in
Tris buffers, particularly with mercaptoethanol present.

> not hurt your template.  It comes down to which is worse,
> contaminating RNAse or mild DEPC treatment.  There are proteinaceous
> RNAse inhibitors available that might be a better choice though.   I'd

The standard protocol for protecting RNA from RNAses during RT uses
placental RNAse inhibitors such as Pharmacia's RNAguard or Promega's
RNasin.

> try a number of methods and evaluate for the best yield of full length
> product.
> 
> I wouldn't expect many enzymes to tolerate more than a few percent
> formamide.
> 
> Regards,
> John Thompson
> Merck Research Labs


Dave

--------------------------------------------
David F. Spencer, PhD
Dept. Of Biochemistry
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca



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