Precision nanoscript2 reverse transcription kit

The TRIZOL (Sigma-Aldrich) method was chosen to obtain total RNA, and precipitate and wash RNA with 85% ethanol to obtain miRNAs. Precision nanoScript2 Reverse Transcription Kit (Primerdesign) and miRNA 1st Strand cDNA Synthesis Kit (Vazyme) were selected to reverse transcription to synthesize cDNA (A260/A280 ratio: between 1.8 and 2.0) for lncRNA and miRNA respectively. The reaction system was configured using ChamQ SYBR qPCR Green Master Mix and miRNA Universal SYBR qPCR Master Mix (Vazyme), and RT-qPCR detection was performed on a 7500 Real-Time PCR system. Cycling parameters: incubation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s, and 72°C for 15 s, and finally extension at 72°C for 10 min. The endogenous control of LINC00943 and miR-196b-5p utilized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6, while the LINC00943 and miR-196b-5p expression were calculated by the 2^−ΔΔCt method. The primer sequences are as follows: LINC00943, F-5’-GATGAACCACCCATGGCCT-3’; R-5’-CTTCCAGGAATGGAAGCCA-3’. miR-196b-5p, F-5’-ATCCTTCCTAGTCCAGCC-3’; R-5’-ACCTGGCGGCACTCCTTA-3’.

To evaluate the expression of ca1pase, mRNA was extracted using a NucleoSpin Tri Prep kit (Macherey-Nagel) including DNase treatment. RNA concentration and quality were determined via a spectrometer (SpectraStar Nano, BMG Labtech). A subsample of 1 µg RNA was used for cDNA synthesis using the Precision nanoScript 2 Reverse Transcription kit (Primer Design) according to the manufacturer’s instructions. Reverse-transcription quantitative PCR (qPCR) was performed with the PrecisionPLUS qPCR Master Mix kit (Primer Design) containing cDNA (1:5 dilution) and the primer pair (Supplemental Table S1) in a Mx3005P qPCR system (Stratagene, Agilent Technologies). Melting curves were also completed. Primer efficiency was analyzed based on a cDNA dilution series with mean primer efficiency estimated using the linear phase of all individual reaction amplification curves and calculated according to Pfaffl (2001) (link). The succinate dehydrogenase (UniGene Cluster ID Ta.2218) and ADP-ribosylation factor (Ta.2291) genes were used as reference genes to normalize gene expression (Paolacci et al., 2009 (link); Evens et al., 2017 (link)). The normalized relative quantity (NRQ) of expression was calculated in relation to the cycle threshold (CT) values and the primer efficiency (E) of the target gene (X) and the reference genes (N), based on Rieu and Powers (2009) (link): NRQ = (EX)^−CT,X/(EN)^−CT,N.

RNA was isolated using a Savant FastPrep FP12 Ribolyser (Qbiogene, Cedex, France) and RNeasy Mini kits (Qiagen, Manchester, UK) according to the manufacturer’s instructions. For the in vitro lung fibrosis model, RLT buffer also contained Proteinase K (Sigma, Poole, UK) to aid lysis. RNA was reverse transcribed to cDNA using a QuantiTect Reverse Transcription Kit or Precision nanoScript2 Reverse Transcription Kit (Primer design, Southampton, UK) according to manufacturer’s instructions. Real-time quantitative polymerase chain reaction (RTqPCR) was performed using a BioRad CFX96 (BioRad, Watford, UK) or a NanoString (NanoString Technologies, Seattle, USA) system with a custom CodeSet. Primers and TaqMan probe sets were obtained from Primer Design. Changes in gene expression were compared to two or three constitutively expressed housekeeping genes (for human samples UBC/A2 and for rat samples Pgk1/Actab/Hprt1). Data were normalised to the mean of the housekeeping genes, and these data normalised to an appropriate baseline control sample.