I am working on mRNA level quantification of copper responsive genes in Klebsiella in the absence and presence of the said metal. I synthesize cDNA using 2ug tRNA and the perform qRT-PCR using 2ul of 10x diluted cDNA. I am using 16S RNA as reference gene but its CT values come between 6.5 and 8.0 while those for target genes are about 24 in the absence and 16-17 in the presence of copper.
Are these CT values lie in reliable range. I have studied somewhere that CT values should be between 15 and 25 . Is this right? Then what should I do? If I dilute cDNA further then Ct value for target gene in the absence of copper will go beyond Above limit.
Should I use 100 times diluted cDNA for 16S RNA gene and 10 times diluted cDNA for target gene?
If I do so then what about data analysis? Will different dilutions of cDNA (Different amounts of template for reference and target genes) have any affect on data analysis results? I use Pfaffle method for relative quantification. Should I mention some where in calculations about this difference in template amount?
Will it be appropriate or can any one suggest something else?