Hi there,
I'm treating MCF7 for an hour with LPS (1ug/ml) in order to enhance the mRNA levels of IL-6 . I measure the expression via Real Time PCR. The treatment seems to affect GAPDH levels (there are differences between treated and untreated cells). I also tried other commonly used housekeeping genes, such as actin or ywhaz, but I still have issues...Apart from that, the Ct calculated for IL-6 in the treated cells is high - close to 28 or 29 cycles. Any ideas?
I may give it a try, thanks. UBC too, some references suggest using it!
Some of ideas for housekeeping genes could be: hypoxanthine phosphoribosyltransferase (HPRT) or Transcription factor IID TATA binding protein (TBP).
Thank you too, I hope to find a housekeeping which is not affected by LPS treatment. After all, there are many housekeeping genes to be used!
I have already used 18sRNA as a housekeeping (used random primers, because of the non-existence of poly-A tail), but it was not that stable...Actually, it was almost like GAPDH!
Dear Thanasi,
as you must already know, there is no such thing as a universal reference gene when it comes to normalizing qRT-PCR results or Western Blots. A good normalizing gene is the one that doesn't move upon treatment of a specific cell type. That being said, here's some insight:
1) 10.1371/journal.pone.0027592
2) 10.1152/physiolgenomics.00025.2005
3) 10.1007/s13353-013-0173-x
4) 10.14791/btrt.2015.3.1.24
Back to your problem now: GAPDH has been shown to play both metabolism and signaling related roles. It's a well established moonlighting protein, so it has logically lost its popularity as a normalizing gene. So the first thing to do is to look in the literature for a variety of other normalizing genes and get the primers. I would start with five different pairs of them and try them in treated/untreated samples plus a primer pair that corresponds to your positive control - a gene that you already know that is regulated by your treatment in the mRNA level. To my experimence, TBP (also mentioned above) works fine, as well as RPL13A both in the human and the mouse cells that I use, none of them is the MCF-7 though. I would also suggest that both your target and your normalizer genes be in the same amplification scale. You can't really compare gene expression at Ct = 19 and another gene expression at Ct = 29! The differences in fluorescence are enormously high and this is not optimal for your subsequent calculations. So if your target has a Ct of 29 (which is quite high) you'd better not use actin or 18SRNA to normalize it!
Finally, if you get such a high Ct for your target gene in your treated sample, you should look back in your experiment set up (materials, LPS gone bad maybe?), as well as the expression profile of IL-6 in your cells. It would also be nice to test your cells since MCF-7 is a cell line that is quite different from lab to lab (up to 8 different karyotypes have been documented, if I'm not mistaken).
Sorry for the long answer. I hope this helps.
A high Ct number may also be dependent on primers performance... Anyway, I would recommend to use at least two or better three reference genes and then performing geometric mean of the values. However, sometimes reference genes movements can't be avoided for different and also unexpected reasons. I agree that GAPDH, ACTB, YWHAZ, B2M, HPRT, 18S and TBP are the most used and reliable reference genes. I use them alternatively, according to specific experimental set-up. I.e. myc is known to induce GAPDH gene, therefore I avoid using it when performing experiments altering myc biology.
In 2014 I was touched in the same problem. I tried to activate some CYP1 family members mRNA expression in MCF-7 cell line using PAH (benzpyrene etc.). I was surprised that I could not see expected results. I mean CYP1 family mRNA overexpression. That time I was use GAPD as a reference gene. I was sad. I had not an opportunity to repeat experiment. When I changed reference gene from GAPDH to ...CYP1A2 - one of the targets (on the base on Ct variation) I could see CYP1B1 and CYP1A1 overexpression (about 4 fold) and the same regarding GAPDH - overexpression.
Moreover, later I tried SCARNA5, RNU1 and some additional non protein coding RNAs as a reference. It was much better. You can try it. It was quite stable.
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