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[PCB2010]

Hi,

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.

[WSN]

To have your new gene's primers concentration at high (e. g. 900nM) while the actin primers at low (e. g. 250nM).