RNA isolation and gene expression analysis for TSLP, CCSP ans SP_D v1

RNA isolation and gene expression analysis Total RNA was extracted from right lung tissue samples (~0,01mg) with Trizol reagent. RNA was subsequently purified using the Direct-zol RNA min prep kit (Zymo Research), following the manufacturer’s instructions, and quantified with a ND-1,000, NanoDrop spectrophotometer (Thermo Scientific) at 260 nm. 1μg of RNA was used as the template for reverse transcription, following the manufacturer’s instructions (EpiScriptTM Reverse Transcriptase System kit, Epicentre, USA), using random hexamer primers (Fermentas, Thermo Fisher Scientific, MA, USA) and a My Cicle rTM BIO-RAD (Thermal Cycler System, CA, USA). Real-Time polymerase chain reaction (PCR) analysis was performed on an ABI Prism 7500 detection system (Applied Biosystem, CA, USA) using Power SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific). Relative changes in gene expression were calculated by the 2-ΔΔCt method normalized against the housekeeping gene 18S. The amplification efficiency for each pair of primers was calculated using standard curves generated by serial dilutions of cDNA, with all primers used being from Invitrogen (Buenos Aires, Argentina) and detailed below: TSLPfp: 5’-AGAGAAATGACGGTACTCAGG-3’, TSLPrp: 5’-TTCTGGAGATTGCATGAAGGA-3’; 18sfp 5’-ATGCGGCGGCGTTATTCC-3’, 18srp: 5’-GCTATCAATCTGTCAATCCTGTCC-3’; CCSPfp: 5’-GATCGCCATCACAATCACTG-3’, CCSPrp: 5’-CTCTTGTGGGAGGGTATCCA-3’; SP-Dfp: 5’-TGGACCCAAAGGAGAGAATG-3’, SP-Drp: 5’-CATGCCAGGAGCACCTACTT-3’.