Hi!
I'm trying to compare the gene expression of a few genes in the murine subcutaneous tumor, spleen to naive pancreas as a healthy control. Tumor cell line is of pancreatic origin, that's why I'm comparing that to the healthy pancreas.
To normalize the gene expression I initially used b-actin. However, Ct for actin in pancreas is ~10 cycles higher (later) than in the tumor, although when synthetizing cDNA I used equal amount of RNA (3 ug) for all the tissues and loaded then equal amount of cDNA for the reaction. Then I used 18S and Rps18 genes as different housekeeping. They showed me that actin is indeed much higher in the tumor tissues. But the problem is still there: both 18S and Rps18 expression (meaning Ct values) were also higher in the tumor comparing to pancreas. So, looks like there is less overall material in pancreas and that's weird as mentioned earlier I used same amount of RNA for cDNA synthesis.
Even more interesting, same situation happened when I tried to compare protein expression in those organs by western blot. Again, when loaded same amount of total protein, I could see that b-actin is much higher in the tumor compared to pancreas. Although some of the proteins (like hsp90ab1) were comparable in both organs. Meaning, again paradoxically it looked like there is more total material in the tumor, although loaded equally.
So, I'm wondering whether someone may know a good way to compare gene and protein expression in pancreas versus other tissues like tumor.
In addition, are there some tricks about preparing lysates of pancreas ? As apparently pancreas has tons of proteases and that may affect the quality of the total lysate. However, it's not clear to me how that affects gene expression data when RNA is already isolated, checked for quality and normalized per total amount.
Thanks for any suggestions and comments in advance!
Best regards,
Evgenii
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