I am currently working on RNA isolation from lung tissues and furthering it with the synthesis of cDNA followed by qPCR involving certain proteins like iNOS and RAGE. Right from isolation of RNA from lungs to cDNA synthesis has been properly carried out but still the delta delta Ct curve has not been demonstrated properly at the end of the experiment. RNA isolated has been quantified using Qubit 3.0 flourometer and quality has been checked by performing gel electrophoresis. cDNA synthesis is performed using TAKARA kit and gel run has been performed to check quality. qPCR is performed using SYBR Green and the housekeeping gene used is GAPDH. The qPCR fails to demonstrate the delta delta Ct curve and relative concentration properly and henceforth the experiment is a repeated failure. Kindly give your insights...

Thanks