GAPDH CT lower than target gene CT - (Apr/19/2010 )
HI,
My gene of interest (GOI) is reduced in cancer tissues compared to corresponding non0tumor tissues. I next want to check for circulating mRNA expression in plasma and I anticipate a reduced expression compared to healthy dnor etc.
I ran a qPCR to detect circulating mRNA of GOI and the relative fold of GOI in healthy donor plasma is lower than the plasma from cancer. This is what I was expecting. But what's troubling me is that the CT value of GOI is lower than teh CT value of the GAPDH reference gene, which is why I got teh results I was anticipating? Doe sthsi mean that the plama is not of good quality? Shall I be using another reference?
SF_HK on Apr 20 2010, 12:54 PM said:
My gene of interest (GOI) is reduced in cancer tissues compared to corresponding non0tumor tissues. I next want to check for circulating mRNA expression in plasma and I anticipate a reduced expression compared to healthy dnor etc.
I ran a qPCR to detect circulating mRNA of GOI and the relative fold of GOI in healthy donor plasma is lower than the plasma from cancer. This is what I was expecting. But what's troubling me is that the CT value of GOI is lower than teh CT value of the GAPDH reference gene, which is why I got teh results I was anticipating? Doe sthsi mean that the plama is not of good quality? Shall I be using another reference?
Usually reference genes have a lower Ct than GOI, but this is not necessary. GAPDH is just one of many reference genes, and a higher GAPDH Ct compared to your GOI just shows that there's more GOI mRNA than GAPDH mRNA in plasma. It is possible that GAPDH mRNA is not carried outside the cell as much as your GOI. I wouldn't worry about it, as long as your preparations and calculations are correct.
Chris
chrisbelle on Apr 22 2010, 06:43 PM said:
SF_HK on Apr 20 2010, 12:54 PM said:
My gene of interest (GOI) is reduced in cancer tissues compared to corresponding non0tumor tissues. I next want to check for circulating mRNA expression in plasma and I anticipate a reduced expression compared to healthy dnor etc.
I ran a qPCR to detect circulating mRNA of GOI and the relative fold of GOI in healthy donor plasma is lower than the plasma from cancer. This is what I was expecting. But what's troubling me is that the CT value of GOI is lower than teh CT value of the GAPDH reference gene, which is why I got teh results I was anticipating? Doe sthsi mean that the plama is not of good quality? Shall I be using another reference?
Usually reference genes have a lower Ct than GOI, but this is not necessary. GAPDH is just one of many reference genes, and a higher GAPDH Ct compared to your GOI just shows that there's more GOI mRNA than GAPDH mRNA in plasma. It is possible that GAPDH mRNA is not carried outside the cell as much as your GOI. I wouldn't worry about it, as long as your preparations and calculations are correct.
Chris
Hi Chris, thank you for your response. Since RNA yield from serum is always very low, I want to avoid treating my seru, samples with dnase after extracting the RNA. In understand to be able to avoid this step, my primer whousl be intro-exon spanning. Do you know of any website which can check if my existing primers are intro/exon spanning? I know there are websites which privide designers taking the intro/exon spanning but the website that I used (primer 3) for selecting my primers, didn't have that option and I just want to confirm.