Quantitative Real-Time PCR Protocol for Gene Expression Analysis

Total RNA was extracted using the TRIzol reagent (Invitrogen). One microgram of total RNA was used for complementary DNA reverse transcription using the Superscript-III First Strand Synthesis kit (Invitrogen) following the manufacturer's protocol. All real-time PCR reactions were performed in duplicates in a 20 μl volume using SYBR Green Master Mix (Invitrogen) in a Realplex2 Mastercycler ep gradient S thermal cycler (Eppendorf). The PCR program followed for all the primers was an activation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, 54 °C for 1 min, and 72 °C for 45 s. The reaction was finally put to hold at 4 °C. Subsequently, 18S was used as the reference gene for the mRNA levels. Relative change in gene expression was calculated according to the ΔΔCt method (53 (link), 54 (link)).