I am very new to RT-qPCR.
I am performing RNAi experiment as described below.
I have silenced one gene (gene A) with four different siRNAs. I performed triplicate experiment for each siRNA.
I used GAPDH as a positive control to check for siRNA delivery efficiency.
I performed triplicate experiment for GAPDH too.
I used scrambled siRNA as a negative control. I performed triplicate experiment for scrambled siRNA too.
Please I need help on the way I should arrange the RT-qPCR for:
1. Each 3 samples of the 4 siRNAs = 12 samples
2. The 3 samples of the GAPDH siRNA = 3 samples
3. the 3 samples of the scrambled siRNA = 3 samples
4. Non-template control (NTC) = ? samples
I was informed that I should use another house keeping gene such as ACTB that was not silenced to serve as a control
for the Gene A and GAPDH which I have silenced.
How do I peeform QC for the transfection reagents I used?
I would be happy to receive help from everyone.
I would be happy to received used protocols that are same or similar to what I want to do.
Thank you very much,