qPCR: CT value of reference gene
Posted 17 October 2012 - 06:18 AM
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?
Posted 17 October 2012 - 06:22 AM
Posted 18 October 2012 - 09:44 AM
Posted 18 October 2012 - 11:40 AM
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.
I never trust anything that can't be doubted.