Increased Ct values, but why? - (Jun/27/2006 )

Hi all,

I'm hoping that someone here can help me. I've only started doing RT-PCR in the last couple of years, and everything was going just fine until the last couple of months. My Ct values suddenly increased during the course of 2 runs (on the same day). I spent a lot of time trying to troubleshoot that problem, and then the machine (ABI 7700) had a power supply meltdown. Now that is fixed, but my increased Ct values remain.
I had to make a new stock of R1 primer (stored at -80 as a powder), as the old stock had run out. That's when I started getting zero amplification with the working concentrations of my primers. I tried an experiment wherein I changed the primer concentrations, and now the concentrations that work best are 3x higher than the original working concentrations. I'm getting what looks like good amplification, but I'm not seeing the single copy standards until ~70 cycles, whereas before I would see them around 45 cycles. This problem is driving me nuts, as I don't know how to fix it.
I've tried talking to ABI tech support, and they suggest that I have an inhibition problem. So I've made new standards, I'm using a fresh new bottle of water, new aliquot of probe, newly aliquotted primers... but it hasn't made any difference.
As my positive control, I've been using B-actin, and there seems to be no inhibition in that. So obviously, it can't be the water, SuperMix, or ROX, since they're common to both reactions, right? Also, I don't think it's the DNA in the standards, because that looks good with the B-actin test. Do you have any idea what is causing this problem and/or how I can fix it?
Thank you so much for any help! -Sarah

Hi Sarah,

How old are your stock primers? Why not synthesise new ones to check its not that?

Also, is it possible that your original workign primer conc was miscalculated?

Hi all,

I'm hoping that someone here can help me. I've only started doing RT-PCR in the last couple of years, and everything was going just fine until the last couple of months. My Ct values suddenly increased during the course of 2 runs (on the same day). I spent a lot of time trying to troubleshoot that problem, and then the machine (ABI 7700) had a power supply meltdown. Now that is fixed, but my increased Ct values remain.
I had to make a new stock of R1 primer (stored at -80 as a powder), as the old stock had run out. That's when I started getting zero amplification with the working concentrations of my primers. I tried an experiment wherein I changed the primer concentrations, and now the concentrations that work best are 3x higher than the original working concentrations. I'm getting what looks like good amplification, but I'm not seeing the single copy standards until ~70 cycles, whereas before I would see them around 45 cycles. This problem is driving me nuts, as I don't know how to fix it.
I've tried talking to ABI tech support, and they suggest that I have an inhibition problem. So I've made new standards, I'm using a fresh new bottle of water, new aliquot of probe, newly aliquotted primers... but it hasn't made any difference.
As my positive control, I've been using B-actin, and there seems to be no inhibition in that. So obviously, it can't be the water, SuperMix, or ROX, since they're common to both reactions, right? Also, I don't think it's the DNA in the standards, because that looks good with the B-actin test. Do you have any idea what is causing this problem and/or how I can fix it?
Thank you so much for any help! -Sarah

Also, I have a question for you. I am also trying to measure some genes that are expressed in very low amounts, and may be single-copy.

Have you found generally that you have high standard errors (between samples) with the high Ct values (over 35) for these kinds of genes?
A few people on here have talked about high errors for low copy number genes, and that it affects the reliability of results.