50 mg from frozen renal tissue (stored in RNA later™ solution, ThermoFisher) were processed with a Trizol protocol (93289, Sigma).and stored overnight at −80 °C. Total amount of RNA extracted, RNA integrity (RIN, RNAIntegrity Number) and purity (A260/A280) were measured by Nano Chip® kit in Agilent 2100 Bioanalyzer (2100 expert software, Agilent Technologies, Walbronn, Germany) and ND-1000® spectrophotometer (NanoDrop Tecnhologies, Wilmington, DE, USA), respectively. cDNA was synthesized using a Xpert cDNA Synthesis Mastermix (GK81.0100, Lot. 7E2709A, GRISP, Porto, Portugal) according to the manufacter’s instructions.
For each PCR reaction, 20 µL volume was used containing 2 µL of cDNA, 10 µL of Sybr Green (iTaq Universal SYBR Green Supermix 1725124, Bio-Rad, Hercules, CA, USA), 0.4 µL of each primer (listed inTable 1 ) and 7.6 µL of autoclaved DEPC water. Gene expression was normalized with GeNorm algorithm, where gene stability was attained with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransferase (HPRT). The relative expression ratio of each of the target gene was computed on the basis of ∆∆Ct (2−∆∆Cp) values.
For each PCR reaction, 20 µL volume was used containing 2 µL of cDNA, 10 µL of Sybr Green (iTaq Universal SYBR Green Supermix 1725124, Bio-Rad, Hercules, CA, USA), 0.4 µL of each primer (listed in