Neutrophil gelatinase-associated lipocalin as a potential novel biomarker for ventilator-associated lung injury

Xiao, Rui; Chen, Rongchang

RT-qPCR. Total RNA was extracted from frozen lung samples using TRIzol reagent (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocols. RNA was quantified by measuring absorption at wavelengths of 260 and 280 nm using the NanoDrop system (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). and stored at -70˚C until required. A total of 1 µg total RNA was reverse-transcribed to cDNA using the AffinityScript QPCR cDNA Synthesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA), and RT-qPCR was performed using the Brilliant II SYBR-Green QPCR Master Mix kit (Agilent Technologies, Inc.) under the conditions as follows: preheating at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 20 sec and 72˚C for 10 sec. The primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and the sequences were as follows: Forward, 5'-CCC TGA ACT GAA GGA ACG-3' and reverse, 5'-TTG GTA TGG TGG CTG GTG-3' for NGAL, and forward, 5'-CAT CCG TAA AGA CCT CTA TGC CAA C-3' and reverse, 5'-ATG GAG CCA CCG ATC CAC A-3' for β-actin. PCR reactions were performed on an iQ5 Multicolor Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Gene expression was measured in triplicate, quantified using the 2 −ΔΔCq method (15) and normalized to the β-actin internal control.