RT enzyme inhibited by NP-40 and Tw20 ???

Pavel Sova ps44 at COLUMBIA.EDU
Fri Jun 10 17:13:24 EST 1994


***Is Reverse Transcriptase inhibited by NP40 and Tween20?***

Hi molecular biologists of the world,

I encountered following problem during an experiment in which I need to 
use RT-PCR procedure.

The samples in which I expect my RNA are obtained by immunoprecipitation, 
because I am studying presence of HIV RNA in complexes precipitated by 
antibody (and Prot A-Sepharose carrier). I have no idea how much RNA is 
there, but it will be probably very small amount.

The samples of immune complexes were processed by Proteinase K lysis in 
presence of 0.5% NP40 and 0.5% Tween 20; I didn't phenolize neither
precipitated RNA because of fear of loosing most of my RNA. I am not much 
concerned about DNA, because at two points during the whole procedure, I 
DNased my samples.

For RT PCR, I am using Gibco-BRL Superscript Preamplification Kit; where 
for RT part of reaction I intend to use 5 ul in 20 ul total. That dilutes 
both detergents to 0.125% concentration. And here is my question: ***Will RT 
enzyme be affected by this concentration of detergents or killed 
altogether?***

Actually, in that matter I phoned the technical help at Gibco-BRL (Life 
Technologies, now) and they told me that SDS kills the enzyme at very low 
concentration (not surprising), but with NP40 and Tween20 they do not 
have any data (excetp that storage buffer for RT has 0.01% or so NP40). 
Then I used their positive control RNA sample, which I supplemented with 
Tween 20 and NP-40 both to 0.125% concentration and used for the whole 
procedure (followed by PCR) such as I would do with my samples (except for 
different primers). To my surprise, I got positive signal in parallel 
without RT enzyme; and a phone call to G-BRL revealed that their positive 
control RNA is contaminated by DNA from vector they use for in vitro 
transcription to get RNA. Their positive RNA is SUPERpositive ;-). She 
suggested to lower amount of input RNA, which didn't help much -- I am 
still geting positive signal. In summary, after laborious experiments 
with their RNA I still don't know the answer on my question: Does the 
0.125% concentration of NP-40 and Tween 20 inhibit Reverse Transcriptase 
reaction? Because I am getting tired of doing many pre-experiments inside 
of my huge experiment, I am turning to you, expert public on Internet. 
Can anyone answer my question?

Thanks in advance,
Pavel

I will post a summary.

+-----------------------------------------------------
 Pavel Sova                          e-mail:
 Molecular Virology Laboratory       ps44 at columbia.edu
 Columbia University
 New York                            
+-----------------------------------------------------




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