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Settings op a RT-PCR, What is my next step


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#1 Blackeyed

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Posted 07 December 2009 - 05:52 AM

Hey everyone,

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
Greetings

#2 JonBio

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Posted 10 December 2009 - 07:51 AM

Blackeyed,

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!




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