Amplify cDNA for qRT-PCR? - (Oct/27/2009 )

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!

miss montana on Oct 27 2009, 09:12 PM said:

One vital thing to consider is the stability of RNA! RNA degrades more rapidly than DNA and is not stable for very long, even at -80 unless it is stored in a buffered solution, so you need to ensure it is appropriately stored or process it before it becomes unusable.

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.

hey these kits do work....infact you can even do a whole genome microarray with as low 500pg of RNA using the amplification procedure of these kits...you can get as high as 10ug of amplified product...infact i am plannin to buy one of the same....

Thanks for the input! Which kit have you had success with (company, catalog #)? Thanks!

Hi all,

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!

By pre-amplification kit do you mean a reverse transcription kit, ie. a kit to made cDNA from RNA? We've been using the Qiagen SuperScript II Reverse Transcriptase kits for years before doing real-time PCR. We usually start with 0.5-1 ug RNA in a 50 uL reaction (scaled up from the kits 20 uL specification) but use half the recommended concentration of enzyme since it's kinda expensive, and we use a lot.