6 replies to this topic

[miss montana]

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!

[Prep!]

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

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!!!

[k_undertoe]

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.

[Helios]

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....

[miss montana]

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

[RLI12]

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!

[biznatch]

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.

Edited by biznatch, 28 May 2012 - 10:02 PM.