qPCR: CT value of reference gene - (Oct/17/2012 )
I'm running qPCR to check for the expression of several genes in a panel of cell lines and I'm using GAPDH as a control. In my qPCR results, the CT value of GAPDH in my cell lines is 36 or 37. I find this very high, I've never had problem in the past. In my cell lines the CT value of the GAPDH is usually around 16-20. The CT values of my targe genes is between 22-36.
I extracted RNA using TRIzol and the 260/280 of the cell line RNA was about 1.8-1.9. The only mistake I made was that I vortxed my DNaseI for a few seconds prior to DNAse treatment of my RNA. Other than that I cannot understand why the GAPDH CT value is so high.
I'm extracting RNA from cells grown in a 10cm petri dish. I wash teh plate with PBS, and add 1ml Triolz and incubate for 5 mins at room temperature (inside the tissue culture hood) and then scrape the cells into an eppendorf? Maybe its worth mentioning that I reverse transcribe 1ug of total RNA to cDNA and then dilute it 5 times for qPCR.
Where am'I going wrong?
Another point to mention is that when I was running my qPCR (ViiA 7 Real Time PCR ) , ued a 0.2ml 96 wel plate witha total reaction volume of just 10ul. The 96 well plate hs aminimum capacity of 20ul.
Those GAPDH values are worryingly high, it should be around 16-20 ideally. I don't think it could be a technical reason. Did you mean you have had normal results with the same cell lines and the same primer pairs before?
Vortexing Dnase can mean you had actually no Dnase treatment. High DNA content can block qPCR, probably even RT. Try to find out if you have DNA contamination in your RNA and in that case repeat DNase treatment with intact enzyme.
RNA 260/280 ratios can vary depending on pH, but usually for RNA is around 2, 1.8-1.9 looks more like DNA ratio.