RNA extraction, reverse transcription, and PCR

Tissue was homogenized and frozen in Trizol reagent (Invitrogen) and stored at −80 °C. Total RNA was extracted following the manufacturer’s instructions. 1 μg of RNA was reverse transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. Synthesized cDNA was stored at −20 °C. The Left primer for RT-PCR is 5′-ATGACATCACCCTTCCAGCA-3′; Right primer is 5′-GGTGATTGGGTGTGTTGTCC-3′. PCR amplifications were carried out in a total volume of 50 μl using specific primers and Advantage2 Polymerase Kit (Clontech). The Left primer for Real-time PCR is 5′- ATTGATGAGTGTGTGAGCGC-3′; Right primer is 5′- CAGTTGATGCCACTGAAGCC-3′. The Real-time PCR was carried out as previously described³⁸.