Mir 106b 5p

Total microRNA was extracted and purified using the miRVana miRNA Isolation kit according to manufacturer’s instructions. RNA (5 μl) was converted into cDNA using the MicroRNA Reverse Transcription (RT) kit and specific RT primers (Thermofisher Scientific). Total RNA (500 ng or 1 μg) was reverse transcribed into cDNA using qScriptTM cDNA SuperMix (Quanta Biosciences, Beverly, MA). cDNA from cells was mixed with PerfeCTa SYBR Green FastMix ROX (Quanta Biosciences, Beverly, MA) or Taqman Universal Mix II No UNG plus specific PCR primers; AKAP12 (Qiagen, Germantown, MD), and TaqMan Assays [miR-106b-5p (Assay # 000442), miR-302b-3p, (Assay # 000531), miR-520e (Assay # 001119), miR-342-3p (Assay # 002260), miR-186-5p (Assay # 002285), miR-885-5p (Assay # 002296)] for qPCR using the Step Up Real PCR system (Applied Biosystems, Waltham, MA). Relative expression of mRNA and miRNA was normalized to GAPDH and snoRNA U44 expression and calculated using the 2-∆∆Ct method. Experiments were repeated three times and in triplicate.

Total RNA was extracted from plasma using TRIzol reagent (Ambion; Thermofisher Scientific, Inc.). Total RNA obtained from plasma was transcribed to cDNA using the TaqMan® MicroRNA Reverse Transcription kit (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.), and qRT-PCR amplification with TaqMan™ Universal MixII (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.). U6 was used as an internal control. All primers (U6, miR-486-5p, miR-106b-5p) corresponding to miRNAs were bought from Applied Biosystems (Thermo Fisher Scientific, Inc. Cat. No. 4427975, 4427975, 4427975). The expression of SP-D were detected by SYBR Green system and normalized with β-actin. The primers were as follows: SP-D, sense 5’-GGGAGAAGATTTTCAAGACAGC-3’ and antisense 5’-CCTCTGTCTTGGAATCAGTCAT-3’; β-actin, sense 5’-GCGGGAAATCGTGCGTGAC-3’ and antisense 5’-GGAAGGAAGGCTGGAAGAG -3’; qRT-PCR analysis was performed using an ABI Prism 7500 Sequence Detector (Applied Biosystems, FosterCity, CA, USA), and calibrated by using the 2^-ΔΔCT method.
ELISA analysis Plasma of Tibetan healthy people and COPD patients were subjected to ELISA analysis for their concentration of SP-D. SP-D ELISA kits from Bioswamp (Wuhan, Hubei, China) were used according to the manufacturer's instructions.