I am working on RT PCR for Tuberculosis. I want internal amplification control for my RT PCR. 18s rRNA is one of the house keeping gene. Can we use the same for PCR internal control ? Can it also clear the state of DNA isolation form Sputum?
I would not use 18S, that is if you want to test clinical samples. Reason is that all clinical samples will contain some 18S DNA, but the amount of 18S will be highly dependent and thus vary between two samples. Thus if you see Ct 28 in one sample and Ct 38 in the other is than the second sample inhibited or did the first sample contain a lot of human DNA.
I would always use some target that is very unlikely to be present in your samples and spike your sample with this material. You could e.g. use a rare deep-sea microbe, a specific non-human viral/bacterial organism, or plant material (unlikely to be present in your sample) and use a specific gene from them. If you always spike your sample with fixed amount of this you can predict the Ct of this (and choose it to be Ct 25 and that would definitely interfere with a weak (Ct
It would be better to use GAPDH as IC instead of 18S. There are some reasons: more stable and uniform transcription in different tissues, less expression of this gene than 18S (lesser copy number of GAPDH RNA - lesser influence to taget gene PCR).
We use the GAPDH as an internal control and we have a good experience with it.
DNA from sputum contains mix population of cells and bacterias. I would recommend to use a combination of 3-5 house keeping genes to take geometric mean to use as normalization factor. You can select about 10 potential HK genes and then use GeNorm method to select the top three HK genes.
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