When puting a real time PCR, parameter CT, which means threshold cycle, is used. What does it mean really? according to wikipedia "The number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle (Ct)" could anyone put it in conext?
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Realtime PCR uses a fluorescent dye which binds to double-stranded DNA and thus allows to measure the growing amount of DNA by each cycle. The DNA added to the reaction also binds the fluorescent dye and makes some part of the fluorescent background. When the PCR reaction starts, it takes a while until enough DNA is synthesized to go over the background and to be able to reliable distinguish the signal from the noise. I think this becomes clearer, when you look at the image below (from the NIH):
The Ct is the point where the signal can be distinguished from the background when it enters the exponential grwoth phase. The no template control stays below this threshold.
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$\begingroup$ and when you make the calibration curve does it really matter which range of dilutions you choose or does it have to be a logarithmic serie? besides, when you get your results for different CTs, which is the criteria when choosing the correct dilution? $\endgroup$– KatzMar 14, 2014 at 11:15
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$\begingroup$ If you know the concentration of your standard exactly, you could do only one reaction. However it is better to do at least a few dilutions to see if your dilutions are correct and to test if your standard is amplified linear (meaning 10x more standard gives an accordingly higher signal). I would always choose a dilution which is as close as possible to your gene of interest. I mostly avoided absolute quantifications if possible, since relative quantifications usually where sufficient for us. $\endgroup$– Chris ♦Mar 14, 2014 at 12:28
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$\begingroup$ what do you mean with " I would always choose a dilution which is as close as possible to your gene of interest."? $\endgroup$– KatzMar 14, 2014 at 15:08
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$\begingroup$ That I use the curve (when I make more than one) which is nearest to my sample of interest. This way you avoid to skew you calculation by non-linear amplifications. $\endgroup$– Chris ♦Mar 14, 2014 at 20:33
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$\begingroup$ and when i use the most diluted concentrations i often get fold changes really diverse when comparing to the more concentrated. Is there any criteria i could follow to discard those results? $\endgroup$– KatzMar 17, 2014 at 14:52