Hi all,
I recently ordered and began culturing a new cell-line that are meant to be a canine line. I was interested in testing their expression of a particular gene, and of course compared this with a common house-keeping gene. I ran qPCR this morning, and gene expression of both my housekeeping gene and my gene of interest are undetectable, despite using canine primer probes.
My colleague believes these cells I have been growing may not be what they are advertised to be - they grow extremely fast (faster than HEK293 in my experience). I will be testing the cDNA I made with both human and mouse primer probes tomorrow to see if these primers work. However, I'm concerned I have done something wrong - I am wondering if maybe the cDNA reaction was set-up wrong? I'm not so sure if this is the case, I check everything off as I go... unless there is something wrong with the heating block... How can I test my cDNA before setting up my qPCR reaction tomorrow? Should I make more cDNA from my RNA? Or, should I go ahead and set up qPCR?
Is there any explanation other than these cells being the wrong species? Perhaps I used the wrong program when doing qPCR? Any advice is helpful - thanks!
Oooh this is an interesting one. There's quite a bit here.
Firstly - I don't think you can assume that if mouse or human primers DO work, that means the cells are human/mouse and not canine. You could check the primer sequences against the dog genome with BLAST and see how similar they are.
Second - Sanity check your reaction setup. How much RNA did you put into each cDNA synthesis, and also what kind of primers and how much? How much cDNA template did you put into your qPCR?
Third - yes you can check if your cDNA synthesis worked. Check the concentration using Nanodrop or Qubit. If it's less than about 20ng/uL, your reaction failed. If it's more than that, run it on a 1.5% agarose gel and see what the size distribution looks like. You should see a broad smear from ~50nt up to ~1000nt - if it's very skewed towards low molecular weight fragments, the problem is that your RNA was degraded.
I would avoid wasting further RT or qPCR reagents until you know if your cDNA synthesis worked and what the size distribution is like.
Also - unorthodox suggestion but it just might work - would any of your canine-specific primer sets detect canine gDNA? If so - does anyone you work with have a pet dog? You would be able to extract a modest amount of canine gDNA from a cotton swab vigorously rubbed on the gums of any dog. This would at least let you check whether your canine-specific primers do recognise the dog genome. (If they are mRNA specific primers, you're out of luck here.)
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