
Amplify cDNA for qRT-PCR?
#1
Posted 27 October 2009 - 06:42 AM
Has anyone successfully used a cDNA amplication kit before doing real-time PCR? I have some RNA left by a former lab member from a rare population of mouse cells. We need to run the samples to look at a few genes, and I definitely won't have enough. Thoughts?
Thanks!
#2
Posted 28 October 2009 - 09:28 PM
Hi All,
Has anyone successfully used a cDNA amplication kit before doing real-time PCR? I have some RNA left by a former lab member from a rare population of mouse cells. We need to run the samples to look at a few genes, and I definitely won't have enough. Thoughts?
Thanks!
It should work!!! or else they will be broke (the manufacturers)
Or you can try on a less precious RNA sample and decide for yourself if it solves your purposes!!!
Cheers!!!
#3
Posted 29 October 2009 - 09:15 AM
A possible consideration for you is to make sure the pre-amp kit is compatible with your downstream qPCR reagents; i.e. make sure the concentration of salts, enzyme etc are not going to interfere with your qPCR stuff. Also, if you are going to use gene specific primers or oligo/random to prime you may need to remove those prior to your qPCR and that can lead to loss of template too. I have had incompatibility happen where the pre-amp had SO MUCH salt in the buffers and such that I had to run a PCR mini-spin purification, which basically put me back to what my starting amount was after you factor in the loss for the column. Just a note of caution.
#4
Posted 01 November 2009 - 12:51 AM
#5
Posted 01 November 2009 - 06:37 AM
#6
Posted 17 May 2012 - 07:01 AM
I have the same problem...any kits that you can recomment me?
I have seen that Roche has an RNA pre-amplification kit but it is now discontinued in the UK. NuGEN is the only possibility that I have found but it is very expensive so I would like to hear some opinions first.
Thanks in advance!
#7
Posted 28 May 2012 - 10:01 PM
Edited by biznatch, 28 May 2012 - 10:02 PM.