Hello!
For a qPCR experiment I have to compare samples from a rat cell line with human samples using TaqMan various assays.
I am currently looking for a set of good primers targeting a common housekeeping sequence between my samples to normalize the DNA inputs.
Does anyone has suggestions of papers/assays that could help me doing that?
Thanks in advance!
Hi,
If you are using Taqman assays, you do not need sequences, just order the assay(s) to target the gene you are interested in.
If you mean which is the best housekeeper to use, the list is very large but there are many papers and recommendations out there, just google 'best housekeeping genes' and you will find plenty in all species.
As with all qPCR experiments, don't rely on just one housekeeper - if that happens to be unstable you may not see the results you are looking for. We always use at least 3 and test them for stability before picking which to use.
Hi!
My goal is to test my samples from two species with the same assay. So I can't use a classical commercial one since they usually don't mention the exact region of the primers/probe (which should be highly conserved accros my species).
Maybe my question was not clear enough, sorry.
Regarding using several housekeeping genes that's of course my plan.
I see.
In that case, you could get the human sequences of some common HK genes from UCSC and BLAT them against the rat genome, which will give you regions of conservation.
You can then use the conserved regions to put into the ThermoFisher Custom Taqman Design tool and this should give you a custom assay that will work on both species.
If you want to avoid a DNase-treatment step then assuming you have good conservation around an exon-exon boundary, you can always 'in-silico' splice the exons and use that sequence to design to and assign the boundary as the target, meaning that your assay will only bind and give signal for mRNA.
If you have reasonable conservation but some bases different, as long as there aren't too many you can replace them with 'n' and the tool will avoid them when designing.
Hope that helps!
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