Taqman real time pcr assay

RNA was extracted by using TRIzol reagent (ThermoFIsher Schientific) or RNeasy Plus Micro Kit (QIAGEN). Complementray DNA synthesis was performed according to the manufacturer’s instructions (qScript™ cDNA Synthesis Kits, Quantabio). Taqman real-time PCR assay (ThermoFisher Scientific) or SYBR green master mix (Applied Biosystems) was used for detecting gene expression. The relative mRNA expression was normalized to housekeeping gene expression (β2-microglobulin). Taqman probes and SYBR green primer sequences are list in Supplementary Table 4.

To extract RNA, cells were harvested, washed with phosphate-buffered saline (PBS), and lysed in RLT Plus Buffer (Qiagen, #1053393), followed by extraction of RNA using the RNeasy^® Mini Kit (Qiagen, #74106) according to the manufacturers’ protocol. The extracted RNA samples were quantified by using NanoDrop™ 8000 Spectrophotometer (Thermo Fisher Scientific) or NanoPhotometer^® N120 (Implen). For gene expression analysis, complementary DNA (cDNA) was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, #4368813) followed by qPCR (quantitative reverse transcription polymerase chain reaction) using TaqMan^® real-time PCR assay (Thermo Fisher, #4331182) for the following genes: COL1A1, COL3A1, COL10A1, ACTA2, and CTGF. The ΔΔCt method was used to calculate the relative expression levels using glyceraldehyde-3-phosphate dehydrogenase as the housekeeping gene. In brief, the Ct values were normalized to the housekeeping control gene GAPDH (delta CT value). The fold change was calculated with the 2-ΔCt/ΔCt equation, which means that each delta Ct value was normalized to an untreated control (medium control).

Total RNA was extracted using TRIzol reagent (ThermoFisher Scientific) and reverse transcribed using cloned Avian Myeloblastosis Virus reverse transcriptase (ThermoFisher Scientific). For qPCR, Taqman real time PCR assay (ThermoFisher Scientific) or SYBR green master mix (Applied Biosystems) was used for gene expression analysis. Gene expression was normalized to housekeeping gene expression (β2-microglobulin). The relative gene expression was calculated by the change-in-threshold (2^−ΔCT) method. All experiments were performed in duplicate in two independent experiments and results are presented as standard error of means of biological replicates. Taqman probes and eRNA primer sequences are listed in Supplementary Table 4 and 5.

Total RNA from cell lines or mouse bronchoalveolar lavage fluid (BALF) was extracted with QIAzol lysis reagent (Qiagen, Germantown, MD, USA); total RNA from mouse lung tissue was extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized with the high-capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. qRT-PCR was performed using a 7500 Realtime PCR system (Applied Biosystems, Foster City, CA, USA) with the Taqman Realtime PCR assay (ThermoFisher Scientific, Waltham MA, USA) according to the manufacturer’s instructions. Primers: il6 (Mm00446190_m1, FAM), il12a (Mm00434165_m1, FAM), il12b (Mm01288989_m1, FAM), ifng (Mm01168134_m1, FAM), ifit1 (Mm00515153_m1, FAM), mx2 (Mm00488995_m1, FAM), oasl1 (Mm00455081_m1, FAM), irf7 (Mm00516793_g1, FAM), ifna1 (Mm03030145_gH, FAM), and 18s rRNA (4319413E, VIC).

For real-time PCR, total RNA was extracted using TRizol Reagent (Thermo Fisher Scientific) followed by reverse transcription using SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). mRNA level was quantified by TaqMan Real-Time PCR Assay (Thermo Fisher Scientific) using the probe Hs00227713_m1 against αTAT1 and GAPDH as the internal control.