Strand cdna synthesis kit

The lung tissues were isolated from the mice with different treatments. The cells were collected after normoxia or hyperoxia exposure. For the detection of miRNA expression, total RNA was extracted using a Total RNA Extraction Kit (Synthgene Biotech, Nanjing, China) following the manufacturer's protocol. RNA quantity and purity were measured by a NanoDrop spectrophotometer (Thermo Fisher, Wilmington, DE, USA). The cDNA was synthesized using a Strand cDNA Synthesis kit (Takara Biotech, Dalian, China) following the manufacturer's protocol. The protocol was showed as follows: 42°C for 60 min, 70°C for 15 min, and chilling at 4°C. qRT-PCR was then performed using TaqMan™ Fast Advanced Master Mix (Thermo Fisher, Wilmington, DE, USA) with the following procedure: 95°C for 5 min, 95°C for 15 s and extension for 1 min at 60°C for 40 cycles, and 60°C for 5 min. U6 and GAPDH were used as an internal control. The data were normalized by the U6 or GAPDH with the 2^-ΔΔCT method [21 (link)]. The primers are the following: miR-21-5p: forward 5′-GTCAATAGCTTATCAGACTGA-3′ and reverse 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCA-3′; U6: forward 5′-CTCGCTTCGGCAGCACACG-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′; PGAM5: forward 5′-GTGCCACTGGATTAGGGCAA-3′ and reverse 5′-GGGACTTCTCTGACCAGGCT-3′; and GAPDH: forward 5′-CAGGACCTCACTCATTGCCC-3′ and reverse 5′-GACGGACACATTGGGGGTAG-3′.

Real-time quantitative PCR (RT-qPCR) was performed as what we did previously.^{44 (link)} Extracted RNAs were transcribed to cDNA using primer Uni-12 or random primers by Strand cDNA synthesis Kit (Takara, Cat # 6210 A). The cDNA was then amplified using gene specific primers (Supplementary Table 5) for DI-PB2, DI-PA, DI-PB1, CD2100 and CD3600 using SYBR Green I Master (Takara). For quantitation, 10-fold serial dilutions of standard plasmid equivalent from 10¹ to 10⁶ copies per reaction were used to generate the standard curve. RT-qPCR was performed using LightCycler^® 96 system (Roche, USA).

Total RNA was isolated from the liver using TRIzol reagent (Catalog No. 15596018; ThermoFisher) and then transcribed to cDNA using a Strand cDNA Synthesis Kit (Catalog No. 6210A; Takara). RT-qPCR was performed with LightCycler 480, and technical triplicates using TB Green reagent (Catalog No. RR420A; Takara). The expression levels were calculated with the 2^^(-ΔΔCT) method, and the CT values were normalized using Gapdh as a reference gene. The target genes were Thbs1 and Osgin1.