Here I have 36-well Rotor gene 6000 cycler and I want to do relative quantitation via two step RT-qPCR - I mean, first I make RT reaction, and then use the product for qPCR.
It works without ROX.
I have some brief manual of the cycler but it doesn`t say much about how to analyze my results and how to optimize the reaction.
I downloaded several manuals, I continue to read, but I have some questions:
1. Should I prepare standard curves for any of the primer pairs with different cDNA concentrations?
2. I read that I have to optimize the concentration of the primers for to avoid dimerization - can I do this by running several NTCs with different dilution of primers or I have to sacrifice some of my RT product? I ask this silly question because the NTCs of some primers are fine, but others have some peaks.
3. What is better - to have separate annealing and elongation step or to unite them? The protocol of the kit includes separate steps - is that ok?
Edited by Nephrite, 17 April 2018 - 09:12 PM.