Large variation in replicates in samples with low gene copy by methyLight. - (Oct/24/2008 )
HI All,
I have used MethyLight technology for a while. When I tried to use it for detecting low copy methylated genes from urine, I encountered the problem of large variability in triplicates. One well of the triplicate may show a Ct of 38, another 39, while the third one of undetectal. The worse is seen in those samples with one of triplicate with a Ct of 38, and the other 2 wells of undetectable. This problem is most likely caused by low gene copies in the samples. Unfortunately, it's not possible to increase the initiate DNA input into PCR mix due to limited DNA available. I'll be very much grateful if anyone could suggest how to quantify those samples, if we are not ready to sacrifice these quantitative data into qualitative data, i.e., positive vs. negative.
Some people said that rPCR results >37 are not reliable any more. However, Narayan Shivapurkar and coauthors could detect genes in linear relation until cycle 44 (Cancer letters, 2007; 247: 56-71). in fact, the first standard point in their standard curve construction started with cycle 36, with serial dilution, their most diluted standard point (SD6) reached Ct 44. In our lab, the Ct of the most diluted standard point never reached so high Ct, still less the huge CV in replicates for those very diluted standard points. Any tips how to make the last few standard points (I mean more diluted SD points) visible with reasonable CV in replicates?
Help me please! Thanks in advance.
sounds a bit dodgy to me.
your incosistencies between replicates and high Ct's suggests you are not amplifying anything.
because of your limiting samples would it be possible to preamplify your samples with a round of normal unbiased bisulphite PCR prior to your methylight quantitation?
Nick
your incosistencies between replicates and high Ct's suggests you are not amplifying anything.
because of your limiting samples would it be possible to preamplify your samples with a round of normal unbiased bisulphite PCR prior to your methylight quantitation?
Nick
Hi, Nick,
Many thanks for your suggestion for a nested rPCR for our samples. We'll try it soon.
I'm a bit puzzelled about your 1st comment. We used primers and a Taqman probe in our MethyLight. Is it still possible that a Ct of 38 means false positive signal? I thought that this is supposed to happen more easily in Syber Green method. Your further explanation will be appreciated.
rPCR7
looks like you have really low template amounts with a CT of 38 and the inconsistency.
TaqMan certainly has increased specificity over SYBR green.
However, if your sample has low template concentrations and you are using say 1uL for PCR, it is almost digital PCR because you could be taking up only one or two molecules of the template of interest, hence the incosistency, because in one time you could take up the template but not in others.
Hope that was clear. 
Nick
Hi, Nick,
Thanks for the further explanation. Yes, fully agree with you to it. Poisson distributions of the targets might be the major cause. We'll try something else to enhance the assay sensitivity.
Regds,
rPCR7.
That is exactly what I was getting at. Couldn't find the words at the time
Nick