making your own qPCR kit! - (Aug/25/2016 )

Dear All, 

we came to a point when applying qPCR in-house protocols, in our lab, 

to need to generate our own exogenous positive controls and internal controls too.

we are dealing with viruses to be detected in animal tissue samples.

what's the best internal control -as from your experiences- to be used ? 

and we need to develop a protocol in which we detect the "pathogen" and the "internal control"

in the same reaction,

i.e : to put the primers and probes for both the pathogen and the internal control 

all in the same tube ... 

making it easier for the work load.

you know any guides i should follow in this regard ???

thanks in advance,

nightingale

So your internal control is just to make sure the reaction is working or that RNA/cDNA was prepared? If so, it would be best to go for a common house-keeping gene such as GAPDH, as these amplify pretty easily and have well established conditions.

Do your animal samples come from different species, or all from the same species? If they are from the same species, this becomes easier... if different species you will need to design degenerate primers, which can work, but are tricky to optimize.