Punica granatum L. Leaf Extract Attenuates Lung Inflammation in Mice with Acute Lung Injury

Pinheiro, Aruanã Joaquim Matheus Costa Rodrigues; Gonçalves, Jaciara Sá; Dourado, Ádylla Wilenna Alves; de Sousa, Eduardo Martins; Brito, Natilene Mesquita; Silva, Lanna Karinny; Batista, Marisa Cristina Aranha; de Sá, Joicy Cortez; Monteiro, Cinara Regina Aragão Vieira; Fernandes, Elizabeth Soares; Monteiro-Neto, Valério; Campbell, Lee Ann; Zago, Patrícia Maria Wiziack; Lima-Neto, Lidio Gonçalves

2.3.4. RT-qPCR Assays in the Lung Tissue. TNF-α, IL-6, IL1β, and IL-10 mRNA expression in lung samples was determined by RT-qPCR. Total RNA was extracted by using a RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions in a QIAcube automatic DNA extractor (Qiagen, Hilden, Germany). Samples were treated with DNase (Qiagen, Hilden, Germany) and then reverse-transcribed using 200 U of SuperScript II reverse transcriptase (Thermo Fisher Scientific) to obtain cDNA. The qPCR assays were carried out in 96-well plates using a double-stranded DNA dye (GoTaq® real-time PCR Master Mix from Promega) in a QuantStudio™ 6 flex real-time PCR instrument as previously described. GAPDH mRNA was used as an endogenous reference gene. mRNA relative expression was calculated using the 2 −ΔCt method [11] . Results represent the mean values obtained from two independent experiments, with assays performed in triplicate.