Hi all,
I am trying to detect the levels of RORC in my cells. I am using a probe from Merck and my housekeeping gene is GAPDH. I am getting GAPDH when I perform the qPCR, but no RORC. This doesn't make any sense to me as I am getting an abundance of IL-23 using ELISA, which is induced in response to RORC expression (so surely it must be expressed). My cells are CD14 cells which have been purified from PBMCs isolated from human donors. I culture the cells at a density of 1x10^6 cells/ml for 24 hours before removing the cells and immediately freeze down the pellets and supernatant for further analysis. For cDNA synthesis, I used 400 ng of RNA per reaction. For the qPCR itself, I am using the Roche Lightcycler Nano, I use the Roche Probes Master Mix, along with probes from the Universal Probes Library which are now made by Merck. I even increased the amount of cDNA to 8 ul and still did not get any Cq values.
Can anyone see if I am overlooking anything? Has anyone had any luck detecting this using similar cells/conditions?
You said the amount of RNA used in cDNA synthesis but didn't mention the volume of the reaction. You can try with 1 to 1.5 mirogram RNA in 20 microlitter reaction.
Hi,
increasing of cDNA input is not adviseable as due to the buffer conditions and the RT enzyme it might affect the PCR negatively (I would guess your Housekeeper is not showing an appropriate shift to earlier Cqs).
Coming from another perspective: How stable is RORC expression? In many cases I had this kind of actiations are transient. So it is possbile that you are too late in a time series to see RORC expression when you are at the point that the downstream response is already in place
Sven
- Badges
- Science method