I've used SDHA and YWHAZ as my housekeeping genes in sheep leukocytes for my gene expression study using qPCR.
Although several papers that I've read said that SDHA and YWHAZ are the most stable HKG, even in diseased animals, and you would expect the Ct around 20-22, I sometimes get Ct of 25-29.
I understand that sometimes it depends on the amount of your RNA/cDNA copies (my sample is so small to begin with, I'm estimating from circulating leukocytes). You will get the Ct difference (delta Ct) as your raw data, but the Ct of my target genes were more 'believable' in the sense that the Ct of target genes in that sample is similar to Ct in another sample.
However, since we need to 'normalise' our Ct to the samples' own HKG (Delta Ct) first, then it appears to me it gives false values - the delta Ct is smaller which later when calculate for fold change, there is high response/expression.
If how I explain here is too confusing, here's my example :
sample 1 :
Ct -
SDHA : 22
YWHAZ : 23
IL-6 : 28
delta Ct = 35 - (mean of HKG 22.5) = 5.5
Sample 2 :
SDHA : 25
YWHAZ : 26
IL-6 : 28.5
delta Ct : 28.5 - (mean HKG, 25.5) = 3
Can you see that how I can see the value for sample 2 as misleading? It appears that sample 2 had higher response, compared to sample 1. whereas it is actually the issue with the HKG that had lower Ct values, and therefore giving smaller delta Ct difference.
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