qPCR

Following reverse transcription of mRNA to cDNA, qPCR for relative mRNA expression may be performed using SYBR Green. SYBR Green dye binds to double stranded DNA. SYBR Green PCR Master Mix available through Thermo Fisher contains the dye, PCR reaction buffer, dNTP's and DNA polymerase. You only need to supply the cDNA template plus the forward and reverse primers.

Cycling conditions for qPCR with SYBR Green

95°C for 10 minutes (once)

95°C for15 seconds followed by 60°C for 1 minute (40 cycles)

Endogenous quantitative normalisation control

An endogenous quantitative normalisation control is a gene whose expression is expected to remain stable regardless of treatment etc and can hence be used to calculate the relative expression levels of your target gene of interest. Beta-actin is one example of a widely used endogenous quantitative normalisation control.