Abi7500 quantitative pcr instrument

The qPCR reaction system consisted of 5 µl SYBR Green Premix Taq (cat. no. RN04006M; Monad Biotech Co., Ltd.), 1 µl cDNA, 0.3 µl forward primer (10 µM), 0.3 µl reverse primer (10 µM) and 3.4 µl H₂O. The thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec, 60˚C for 30 sec and 72˚C for 15 sec. Dissolution curve was produced using the standard dissolution curve program (Agilent Aria Software; version 1.5; Agilent Technologies, Inc.). The PCR reaction was carried out on an ABI 7500 quantitative PCR instrument (Thermo Fisher Scientific, Inc.). The relative mRNA expression data were calculated using the 2^-ΔΔCq method (20 (link)). The relative expression value of a gene was normalized against the expression of Actb from control group (WNS) mRNA. The Data was analyzed using the SPSS 22.0 software (IBM Corp). Primer sequences are presented in Table I.

Trizol (Thermo Fisher Scientific) was used to extract the total RNA, and the integrity of total RNA was detected by gel electrophoresis. Absorbance at 260 nm and 280 nm was measured by Nanodrop 2000 (Thermo Fisher Scientific). Next, 1 μg of total RNA was reversely transcribed into complementary DNA (cDNA) by PrimeScript RT reagent kit with gDNA Eraser Kit (RRO37a; Takara Holdings, Kyoto, Japan). Specific cDNA was generated by TaqMan MicroRNA reverse transcription Kit (4366596; Thermo Fisher Scientific) and then specific RT primers from TaqMan MicroRNA Assay were used to generate miR-381. The expression of miR-381 was measured by TaqMan miRNA assays according to the manufacturer instructions (Thermo Fisher Scientific). RT-qPCR was carried out on ABI7500 quantitative PCR instrument (Thermo Fisher Scientific) with SYBR Premix Ex Taq (Tli RNaseH Plus) kit (RR820a; Takara Bio, Shiga, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal reference, 2^-ΔΔCt shows the fold change of the target gene between the experimental group and the control group, where ΔΔCT = Ct (experimental group) - Ct (control group), and ΔCT = Ct (gene) - Ct (internal reference).^{22 (link)} The primers used in the experiment are shown in Table II. All primers are provided by Shanghai GenePharma.

Total RNA was extracted from A549 cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and quantified. A total of 1 µg of RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega Corporation) according to the manufacturer's protocol. The qPCR procedure was conducted utilizing SYBR-Green (Takara Bio, Inc.) on a ABI7500 quantitative PCR instrument (Thermo Fisher Scientific, Inc.) in line with the manufacturer's guidelines. The thermocycling conditions were as follows: 94°C for 5 min, followed by 40 cycles at 94°C for 15 sec, 60°C for 25 sec and 72°C for 30 sec. The sequences of primers used were as followed: MNX1, forward, 5′-GCCTAAGATGCCCGACTTCAAC-3′, reverse, 5′-CGCGACAGGTACTTGTTGAGCT-3′; CCDC34, forward, 5′-ACAGAAACAGGTGCGCTTACC-3′, reverse, 5′-CAGCCGGTCACGTTCTTCTTT-3′; β-actin, forward, 5′-AGCGAGCATCCCCCAAAGTT-3′, reverse, 5′-GGGCACGAAGGCTCATCATT-3′. Gene expression was calculated by 2^−ΔΔCq (26 (link)) and normalized to the housekeeping gene β-actin. All experiments were repeated three times.

Total RNA was extracted with TRIzol reagent (15596026, ThermoFisher) and reversely transcribed into cDNA by PCR Amplifier (Bio-Rad). An RT-qPCR experiment was conducted using the ABI7500 quantitative PCR instrument (ABI7500, ThermoFisher). β-actin and U6 were used as the internal references. The relative transcriptional levels were calculated by 2^−ΔΔCT. The primer sequences are shown in Table S1.