Abi prism 7500 qpcr system

The mRNA expression of autophagy marker LC3 was quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from rat RV and LV free wall tissues using TRIzol (Invitrogen, Carlsbad, CA, USA), and then subjected to reverse transcription into cDNA using a Reverse Transcription Kit (Takara, Dalian, China). The purity of RNA (260/280 nm ratio) was determined spectrophotometrically using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Rockford, IL, USA). Two-step qRT-PCR was used to quantify relative LC3 mRNA by the ABI PRISM® 7500 qPCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green. The relative LC3 mRNA expression was normalized to the level of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) using ΔΔC_T method. Each reaction was carried out in triplicate. Using Primer Premier v5.0 software (PREMIER Biosoft International, Palo Alto, CA, USA), primers for LC3 and GAPDH were designed as follows:

rat LC3 (NCBI accession number: NM_199500.2): 5′-CGAGAGCGAGAGAGATGAAGACGG-3′ (sense) and 5′-GGTAACGTCCCTTTTTGCCTTGGTA-3′ (antisense);

rat GAPDH (NCBI accession number: NM_017008): 5′-TCCATGACAACTTTGGCATCGTGG-3′ (sense) and 5′-GTTGCTGTTGAAGTCACAGGAGAC-3′ (antisense).

Total RNA was isolated from 10 mg each of snap-frozen lung tissues using the TRIZOL Reagent^®(Invitrogen) and quantified using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Rockford, IL, United States) and reversely transcribed into cDNA using a PrimeScript^®RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturers’ instructions. qPCR was performed using the SuperScript III Platinum SYBR Green Two-step qRT-PCR Kit (TaKaRa, Dalian, China) in the ABI PRISM^®7500 qPCR System (Applied Biosystems, Foster City, CA, United States) according to the manufacturer’s protocol. The primers for α7nAChR (GenBank #NM_012832) were 5′-GCAAAGAGCCATACCCAG-3′ and 5′-CAGCAAGAATACCAGCAGAG-3′; GAPDH (GenBank #M17701), 5′-TCCTGCACCACCAACTGCTTAG-3′ and 5′-AGTGGCAGTGATGGCATGGACT-3′. The relative level of α7nAChR mRNA was calculated using the 2^-ΔΔCt method after normalization to GAPDH mRNA.

The total RNA was extracted by Trizol (15,596,026, Invitrogen). The RNA was reversely transcribed into cDNA with the help of a PrimeScript RT reagent Kit (RR047A, Takara Holdings Inc., Kyoto, Japan). The synthesized cDNA was checked through RT‐qPCR with the help of Fast SYBR Green PCR kit (4,364,344, Applied Biosystems, Carlsbad, CA) and ABI PRISM 7500 qPCR system (Applied Biosystems). The 2−ΔΔCt method was utilized to analyse the relative expression of MELK, EZH2 and LATS2 genes with β‐actin as an internal reference. The primer design is described in Table S2.