There are many methods for quantification of gene expression (fold change calculations) through real time PCR such as Pfaffl method, delta ct method, standard curve method. So I want to know which one to use for getting reproducible, consistent, reliable results.
We always had very good results with delta ct method. Both in vitro and in vivo experiments as well as clinical trials.
We always had very good results with delta ct method. Both in vitro and in vivo experiments as well as clinical trials.
It depends on your needs. For routine checking of gene expression for exploratory studies the Delta CT method is the most practical as it allows you to quickly get an idea of the up-or down-regulation of a gene. However, if you're doing a serial study in which your samples and controls are evaluated longitudinally (typical for human samples where is difficult to have all the experimental groups together to be analyzed in a single experiment or in a single plate) then having an internal control (not the same as normalizer, reference gene or housekeeping gene) plus a standard curve that you'll include in every measurement, then would allow you to convert all your data not in fold-changes but in 'copy numbers' and if you manage to collect a lot of samples, this approach is very powerful. - Although it has the disadvantage of having to run a standard curve with every plate using identical standards. For this purpose, getting enough of a good template from the beginning is paramount (linearized plasmid or long synthetic oligo) to be aliquoted.
with my experience I found delta delta CT method is most reliable as we always have a housekeeping gene for normalisation. However you need to check with your treatment which housekeeping gene works best. 18S for plant tissue and actin for animal tissue are most widely used housekeeping gene.
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