Everyone knows that endogenous control is a yard stick for measuring the expression level of our gene of interest in control and treated samples. Do these endogenous controls vary during experimental procedures? If so will it be a good endogenous control? If one faces such a alteration how can one interpret his findings? Please do enlighten me.
Gerson is right on about this.
This needs to be done conscientiously and cautiously, and using the tools available out there for that. Money and time is always an issue finding the appropriate reference genes for each experimental system.
Some might put Santa Claus the Easter Bunny and reference genes (AKA endogenous control genes, housekeeping genes) in the same category: e.g. do they really exist? To which I always point to the quote from the stalwart 2002 J. Vandesompele et al. paper (which includes things that Gerson has just mentioned as well):
From that paper at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126239/
"...Taken together, our data and these studies clearly show that ideal and universal control genes do not exist. This warrants the search for stably expressed genes in each experimental system, and for the development of an accurate normalization strategy..."
Each different system, tissue, sample type, treatment used, sample isolation method, may require different reference genes each time. This is what the reality is for the most part...
Thus, there really is no easy/straightforward answer on this. Further, when using reference genes, apparently you need to find at least 3. And that invariably means sifting through dozens of possible candidates for each different experimental system you engage.
Hello there
Yeah, depending on your experimental conditions and cell type, endogenous controls may vary. There are many papers on this matter, especially on selecting endogenous controls for cancer cells.
If one faces variation in endogenous controls, relative quantification of the target gene will be compromised. Probably the best way out of this is to select a panel of endogenous controls, run your qPCR, and determine the most stable gene(s) across samples. There are many ways to do this, e.g. by using GeNorm or Normfinder.
Gerson is right on about this.
This needs to be done conscientiously and cautiously, and using the tools available out there for that. Money and time is always an issue finding the appropriate reference genes for each experimental system.
Some might put Santa Claus the Easter Bunny and reference genes (AKA endogenous control genes, housekeeping genes) in the same category: e.g. do they really exist? To which I always point to the quote from the stalwart 2002 J. Vandesompele et al. paper (which includes things that Gerson has just mentioned as well):
From that paper at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126239/
"...Taken together, our data and these studies clearly show that ideal and universal control genes do not exist. This warrants the search for stably expressed genes in each experimental system, and for the development of an accurate normalization strategy..."
Each different system, tissue, sample type, treatment used, sample isolation method, may require different reference genes each time. This is what the reality is for the most part...
Thus, there really is no easy/straightforward answer on this. Further, when using reference genes, apparently you need to find at least 3. And that invariably means sifting through dozens of possible candidates for each different experimental system you engage.
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