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:
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!!!
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.