Very basic RT-PCR question

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Fri May 18 04:39:00 EST 2001


Dear Karthik, 

The answer probably depends a lot on the brand and source of 
your RT. Some people suggested in this NG not to use all-in-one 
kits if you have a critical template. In practice, probably 
elongation speed, error rate and average transcript length are 
important parameters. What about the length of the viral genomes 
they have to transcript in "real life"?

You could test the suitability of the RT of your choce simply by 
choosing an appropriate positive controls, i.e. a mRNA of 
sufficient length and known copy number (like the genes used 
for normalization of microarray data) and checking by a 
subsequent PCR, since the performance of the RT seems to me 
being critical for the success of your experiments. (I would 
strongly suggest to include appropriate positive controls for 
ensuring the quality of your library.)

I have been recommended the MMLV RT from Roche as a suitable 
enzyme for critical experiments, and in my experiments, it 
worked well (althogh the transcripts were less than 2 kb). 
Ask Roche for any information you need.

Is there anyone out there who maybe could recommend reading a 
chapter in one of these numerous books PCR? 

And: Never be embarrased to ask questions like that one. The 
only silly questions are those you easily may solve by reading 
standard textbooks or doing a Medline search. Obtaining 
information on these (on a first glance maybe silly looking) 
questions is what makes a lot of experience to everybody of us. 
And in this methods NG, you'll probably find someone knowing an 
answer (and more who are also interesting the answer, but 
either never dared to ask, too, or never thought about this 
issue before). When I got into molecular biology business, I 
had to start almost from scratch (being a chemist) and this NG 
was (and still is) an important source of knowledge and 
discussion for me. 

Stay tuned to this NG!

Wolfgang



> From:          kaghoram at unity.ncsu.edu (Karthik Aghoram)
> Subject:       Very basic RT-PCR question
> Date:          17 May 2001 13:18:40 GMT
> Organization:  North Carolina State University
> To:            methods at hgmp.mrc.ac.uk

> 
> I am almost embarassed to ask this, but here I go anyhow:  Are there any 
> known limits to the range of reverse transcriptase?  One of my colleagues 
> is using a long-range thermo-polymerase to amplify long sequences and 
> build a library, but if the RT "fell off" before reaching the end of the 
> CDS, one is s**ewed, right?  
> 
> I have never been able to get a straight answer to the limits-to-RT
> question.  Thanks in advance for any insight. 
> 
> 
> 
[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
-----
Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
wolfsc at REMOVETHESEANTISPAMCAPITALSibms.sinica.edu.tw
-----

---




More information about the Methods mailing list