Help with high Ct values for Real-Time PCR - (Jan/05/2012 )
I am relativiely new to Real-Time PCR and need some help. I am using mRNA extracted from rat liver that I have reverse transcribed to cDNA. I have used this cDNA before (SYBER GREEN system) on another gene and everything worked fine (Ct around 21). This new gene is giving Ct values around 30 with primers I designed. I would like to get those values lower. I know that I could increase the amount of cDNA but when I do, my reference gene's (B-actin) Ct gets too low (Ct around 15). I would like to avoid this if possible so that I can still use the Pfaffl method for analysis. Or can I use the Pfaffl method on different amounts of cDNA (we're talking like orders of magnitude different).
Any suggestions would be very helpful. Thanks.
To have your new gene's primers concentration at high (e. g. 900nM) while the actin primers at low (e. g. 250nM).