Hello,
I have extracted RNA from the Intestinal Enteroid Cell Line and have measured the expression of multiple genes using RT-qPCR. I tested two housekeeping genes GAPDH and ACTBR both showed good CT values: 18 and 19 *mean values. However, I recently measured their expression using the same primers and dilution factors done in the previous experiment. It was conducted on FH74 cells (Premature infant intestinal cells) However, this time CT values for GAPDH were ~30 and ACTBR were ~28. So it appears that there is expression but not as great as the enteroids. Is it possible that the difference in cell lines would generate that much of a difference for housekeeping genes?
P.S. RNA yield and cDNA yield were higher for the FH74 cells
I plan on rerunning the experiment with higher concentration of primers. To try and trouble shoot the issue. I was hoping to hear your thoughts on the discrepancy of CT values for the house keeping genes.
Yep, no reason why two different cell lines should have the same amount of mRNA molecules (of the transcript of interest) even if their starting RNA and cDNA for PCR was the same. It would be more of a problem if the technical replicates of your cell lines had differing Ct values. This is part of the reason why you have to conduct a reference gene stability study (GeNORM, NormFinder, etc). One thing I can tell you from my experience, never assume a gene is stable in your experimental groups because some similar study in the literature mentioned so, I found that some of the assumed candidates were not so stable during my PhD experiences with RT-qPCR.
What were the 260/230 ratios like for the second set of cells?
Cq values of 28-30 indicates very low levels of expression, and for genes as high abundance as GAPDH and ACTB, this shouldn't be the case.
It sounds like poor cDNA synthesis efficiency to me.
Also "cDNA yield were higher for the FH74 cells" -how are you assessing this?
Hi Rico Carter ,
Different cell types can express different levels of housekeeping genes. Although they usually don't vary wildly, small differences can really change the experimental outcome. In order to circumvent that some protocols use 2-3 housekeeping genes.
Ct values are mostly determined by initial concentration of the template. If your amplification curves are sigmoidal, then it's likely your primers are in abundance and won't influence the Ct values.
"Higher primer concentrations often contribute to mispriming and nonspecific amplification. On the other hand, low primer concentrations can result in low or no amplification of the desired target"
https://www.thermofisher.com/tr/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-component-considerations.html
Hi Everyone, Can Kiessling Ali Ahmed Alawady Dino Santos Matias John Hildyard Thank you for your insight. I am sorry for the delay in response. However, I wanted to give you all an update. After multiple steps of trouble shooting, the issue was with the conversion of RNA to cDNA. The technique was not clean using the FH74 cells. Once, this was cleaned up the expression was similar.
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