Hi, I am working on t24 cell line. Before this, I did RT-PCR from cells that I cultured on 10mm dish. The RNA extraction was done using Trizol method and the nucleic acid concentration was 800ng/ul, with 260/280 = 1.96. The ct value for my GOI was around 22 - 24. However, when I repeat the same method using 24 well plate, 260/280 = 1.95 and nucleic acid concentration = 80ng/ul, the ct value for my GOI is different. It's around 31 - 33. Is this because of the difference in nucleic acid concentration?
Did you adjust the volume of extraction reagents (Trizol etc) such that it remains roughly the same per number of cells grown in either 10mm dish or 24well plate? This way should be able to get similar RNA concentrations at the end. Did you use same concentration of RNA for cDNA synthesis followed by using same amount of cDNA for real time? This will make sure that you get similar Ct values for the same GOI in the same cell type under similar experimental conditions.
Rashmi Pathak ,yes, I used the same concentration of RNA for cDNA synthesis and followed the same amount of cDNA for real time. After your suggestion, I optimized the volume of extraction reagent (Trizol) and the result of the nucleic acid concentration (nanodrop) was different based on the type of well plate/dish used. Amazingly, both Ct values for same GOI is similar! You saved my day! Thank you for your suggestion!
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