Inconsistancy in the CT values for the same samples! - (Jan/24/2007 )

Hello, i am fairly new at the Qpcr experiments, and i am currently facing some difficulties in replicating my CT values for the same samples. Here is the situation ; i have repeated 2 samples in triplicates 3 times and each time the sample has amplified at a different CT value. The Ct values are off by atleast 2 cycles fluctuating up and down.I have kept the concentration of the RNA same all three times. I am completely confused by these reading and would really appreciate any input.
Thanks
Arsu

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Arsu, I have several years of experience with Real Time PCR. It is an outstanding tool to quantitate RNA levels. However, it is very sensitive and requires extremly careful pipeting and sample handling. The first time I tried qPCR I had to repeat it 2-3X before I was able to get consistent results. I have listed some tips below. Although they may be obvious, they are crucial to reproducing results.

Some Tips That May be Useful:

1) Make sure you are pipeting accuratley. Your pipetors must be calibrated. When adding the RNA/cDNA sample, be sure that your pipetor is bringing up the same volume each time and that your samples are well mixed.

2) Check your primers. It is necessary that the primers you are using are specific for your gene of interest. If there is non-specificity present it could alter your results drastically.

3) When handling your RNA samples be sure to keep them on ice and practice RNAse free techniques.

4) Make sure not to cross-contaminate samples. Spin down your tubes before opening the caps.


I hope this helps you somewhat. Good Luck with the qPCR!

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Hi,
I found when i diluted my cDNA 1 in 5 in dH20 (in a 2-step QPCR) this increased the consistency of the Ct values produced.


Incidentally, does anyone know why this is, or who first suggested it.

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Thank you for the quick response. All the things that you have mentioned i have taken in to consideration. Also my replicates have very close ct values on the same plate, and wouldn't the replicates take care of the pipetting error? The problem is that the Ct values fluctuate up and down from plate to plate for the same sample but the replicates remain the more or less very similar in Ct values. The amount of RNA added is also kept same. so i am not sure what exactly is causing this fluctuation.
Once again i really appreciate your response
Arsu

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I would try a few things

1. check that the wells in your machine are not contaminated with fluoresence - although this probably isn't the problem, since your triplicates are consistent, it's a good thing to check anyways

2. what are your reaction efficiencies?

3. are the Ct's going up or down over time? or are they just all over the place? I would suspect stock RNA sample degradation if you see the values creeping up...if they are all over the place, I would guess there's something in your reaction mix that is interfering, inhibiting, or degrading. you may wish to check all the components. perhaps you need new dNTP stock, or new primers?

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Hi!

I am also new with real-time PCR. I work on 16S rRNA gene. I do real time pcr with general 16S taqman probes and primers. They are used by someone else in my lab without any problems. She helped me at the beginning but she's not free anymore. I have two problems ( or many resumed in two part )

I have exactly the problems of different Ct value between my triplicates. I work with plate of 96:
I check my pipetting, try to work with multichannel pipette, and do half-plates to be faster at the beginning.
vortex my samples and stantards, and mix then with pipetting.
work all the time on ice
However it is going worse and worse. Now I have a lot of wells with no amplification at all. The background is increasing. And the curve are chaotics. Could it be that somehow my probe or the primer or not working anymore. I was told different thing: to work in the dark because the probe, And then that it does not matter because there is the quencher, and the time at light is not long enough to affect the result.

Now I do not know what is wrong and what I can try.

When I can compare results I also do not have the same CT number for the same standard between two run. Is it normal? How could results between run be compared? (I always do a dilution series from the standard (E.coli 16S DNA) stock before a run)

Thanks a lot for all the different advices I allready read.

Caroline

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I know I repply to miself...

Someone in my lab has perhaps found one solution. It is still pipetting errors. On her counsel results are better. I would have never guessed it was so sensitive. Her way of loading plate seems to do the difference: first the sample and standard. And then the PCR Mix...

I hope it will work now...

Caro

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Hi,

have you checked if the crossing threshold (or base line threshold) has after every run the same value? When I repeat the same samples in different runs, I adjust the threshold manually. Then I have nearly the same CTs (maximal 0,03 difference) in every run.
Perhaps this will help you

klibi

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Hi klibi!

Thanks, I will try that!

Caro

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