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!
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5 replies to this topic
#2
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Am I me???!!!
Posted 28 October 2009 - 09:28 PM
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!
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!!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!
Cheers!!!
#3
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Posted 29 October 2009 - 09:15 AM
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.
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
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Posted 01 November 2009 - 12:51 AM
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....
#5
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Posted 01 November 2009 - 06:37 AM
Thanks for the input! Which kit have you had success with (company, catalog #)? Thanks!
#6
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Posted 17 May 2012 - 07:01 AM
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!
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!
