analysing qpcr data and plotting standard curve - (Jul/26/2013 )
I once again need your help.
I recently performed qpcr on my cDNA sample.
I isolated rna from my transfected cells and did a two step RT-pcr using random hexamer as a primer to convert RNA to cDNA and used this cDNA as my template in qpcr.
I did three different transfection(WILD TYPE/MUTANT TYPE AND EMPTY TYPE VECTOR TRANSFECTIONS)
In my qpcr i used GAPDH(universal master mix) and for my samples i used gene specific primers and power sybr green probe
i performed my experiment in triplicates with -RT as a control.to know assay effeciancy i made serial dilutions of the cDNA for each(WT/MT/ET),that is concentration starting with 25ng/ul,2.5ng/ul and 0.25ng/ul.
the problem now after performing experiment i want to plot the standard curve (absolute quantification) and calculate the reaction efficiency from the slope. Could you please tell me how to plot this curvea and slope.
I can also see signal for negative control(-RT), this can be due to genomic DNA present in it right?
I am unable to attach the files in here, so if you could relp me i wil forward those files to you guys.
Thanks in advance.
I don't do qPCR very often, but I think you will find that for absolute quantification, you need to have a series of reactions with a defined number of copies (plasmid perhaps) of the target gene.