Hi everyone,
I would like to know that if my normalized gene " 18srRNA" was detected at ct=9 but my interested gene was detected at ct=29, I would like to know that why is the low ct not good for real time PCR detection. Also, why is the recommend the proper ct around 15?
Thank you
Pannapa Pinweha
Low or high Ct is not the issue. The real issue is that your gene amplification is in the linear range for the assay. I usually run 10 fold dilutions (at least 3) to demonstrate that the samples are amplifying according to their concentration correctly. Sometimes when you are amplifying so early (or very late) your amplification does not accurately follow the dilution. With your Ct so low, you should dilute your samples for future runs. I recommend just doing a dilution series to determine the linear range for your assay. This will give you confidence for future runs and probably extend the life of your cDNA pool since you are using less material per run.
Hi Pannapa,
Expression levels of your normalized gene can change according to your protocol and mix configuration.
You can use different dilutions of cDNA for reference genes and genes of interest, as long as all samples are treated the same. This becomes necessary when there is a large difference between expression levels of the two genes. You need to get both genes to fall within the standard curve to validate that they are being measured accurately. Any quantification outside of the standard curve is extrapolation. I often run standard curves at 1:4 dilution series, which means about 2 cycles in between standards (as opposed to 3.33 with a 1:10 series). This allows me to make a thorough standard curve with 5 -7 standard points. If you have sample/standard being detected before 15ct, the concentration of that gene is very high and cannot be measured accurately.
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