I am new at QRTPCR. I tried a lot of reactions by equaling the amount of cDNA concentrations of my samples to 100 ng but i got variable results. My friends suggest equaling the RNA concentrations before cDNA synthesis. At this time I equal RNAs but i think i used small amount? I used 150 ng RNA for total 20 ul RT working volume. It means i used 7.5 ng/ul RNA per RT reaction. I think i should use app. 150 ng/ul right? i couldn't make a standart curve with my cDNAs because the concentration of cDNAs and its dilutions are too dilute?
And i have another question. Is the DNA contamination in total RNA isolation so important? Cant i use in cDNA synthesis?
Thank you for all...