Rneasy plus min kit

The total RNA was extracted from the frozen tissue samples using RNeasy Plus min Kit (Qiagen, QiagenGmBH, hilden, Germany). The purity and concentration of the extracted RNA was determined spectrophotometerically at 260 and 280 nm wave-length (Nanodrop thermoscientific S.N:D015). The cDNA was synthesized from 1 µg of the total RNA using QuantiTect reverse transcription Kit (Qiagen, QiagenGmBH, hilden, Germany) according to the manufacturer´s instruction.

RNA was isolated from lung tissue with the RNeasy plus min kit from QIAGEN 74136. A master mix of RLT plus was made with 10 μL of beta‐mercaptoethanol (from Sigma Aldrich [m6250]) per milliliter of RLT plus buffer (part of the QIAGEN kit). A total of 20 mg of each tissue was then homogenized individually in 600 μL of the mixture per sample. Next, complementary DNA was synthesized with the SuperScript VILO IV kit (11754050; ThermoFisher Scientific). Finally, gene expression levels of ACTB (Taqman Mm00607939_s1), SELPLG (Taqman Mm01204601_m1) were assayed by RT‐PCR with Taqman Gene Expression Mastermix (4369016). Finally, we compared SELPLG messenger RNA (mRNA) levels relative to β‐actin levels in lung tissue homogenates by real‐time PCR in SB and LPS + VILI‐challenged mice.