Settings op a RT-PCR, What is my next step - I`ve got RNA --> cDNA and working primers, what now? (Dec/07/2009 )
I`m a very newbie in the field of RT-PCR and surprise, I`ve managed to isolate my RNA, transcribed it to cDNA and tested my primers in a normal PCR reaction and it all worked.
Now, I want to test my primers in a RT-PCR reaction. But i still got some questions.
How do you design a program for the reaction? I`ve got the Tm`s of my primers and do I just change that one ( I`ve got a GAPDH reaction program) in the program?
Can I test multiple primers during one run?
Do I just than take a somewhat universal Tm for all my primers?
If my primers work on the RT-PCR, can I than measure my samples ( Duplo- Triplo??) and calculate the deltaCt or do I have to perform dilution curves or something?
Thanks in advance
If you're using the delta Ct method, you should check the efficiency of your primers before you can be certain of your RT-PCR results. This is important to be able to compare your gene of interest with your housekeeping gene. Your primers should have 90-110% efficiency and there are many posts on here about how to set up, calculate, etc. Good luck!