Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum > Real-Time PCR and Quantitative PCR Forum
Register Search Today's Posts Mark Forums Read

Real-Time PCR and Quantitative PCR Forum Real-Time PCR and Quantitative PCR Forum


Total RNA, mRNA quantitation by qPCR?

Total RNA, mRNA quantitation by qPCR? - Real-Time PCR and Quantitative PCR Forum

Total RNA, mRNA quantitation by qPCR? - Real-Time PCR and Quantitative PCR Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-29-2009, 10:50 AM
Pipette Filler
Points: 6, Level: 1 Points: 6, Level: 1 Points: 6, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Total RNA, mRNA quantitation by qPCR?



Hello,
I'm assuming there's a fairly fundamental reason why this isn't done, at least I haven't been able to find any informative reference so far...

I want to accurately quantify DNA, total RNA (~rRNA) and mRNA across many, very small samples (mutants and timecourse). DNA, easy enough, but for RNA I don't see why I can't just RT using either random or oligo(dT) primers, and then run the qPCR rxn with random primers (I have random 15mers, Tm very low at 40C though). I don't want to make ANY assumption about 'reference' mRNA here - the conditions are potentially highly dynamic. Possibly I could go for specific primers to the major rRNA species, but that would be a bit of a pain.

Anyone else tried this or know of a reason it would not work?

I've done a single trial run, 10ul rxns with 10-fold dilutions from 10ng down to 10pg, but no take-off whatsoever, nothing on the melt. I might try larger rxns, maybe 20mer primers, higher [primer]. It would be very nice if this would work, but perhaps I'm being too simplistic.

Cheers
Reply With Quote
Reply

Tags
dna , mrna , qpcr , quantitation , rna , rrna , total


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
qPCR NEWS January 2009 - update in available real-time PCR cycler Editor www.Gene-Quantification.info Protocols and Methods Forum 0 02-19-2009 11:04 AM
qPCR Newsletter July 2008 - main focus on qPCR optimisation Editor www.Gene-Quantification.info Protocols and Methods Forum 0 07-28-2008 02:48 PM
qPCR NEWSLETTER - September 2007 - qPCR 2007 talks online Editor www.Gene-Quantification.info Protocols and Methods Forum 0 09-25-2007 12:34 PM
qPCR NEWSLETTER - July 2007 Editor www.Gene-Quantification.info Protocols and Methods Forum 0 07-27-2007 11:04 AM
qPCR 2007 - CALL for scientific contributions Editor www.Gene-Quantification.info Protocols and Methods Forum 0 01-22-2007 02:24 PM


All times are GMT. The time now is 09:27 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12105 seconds with 16 queries