Primer script reverse transcriptase kit

Total RNA from clinical samples or A549 cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After measuring the purity at a wavelength of 260 nm and concentration of RNA using NanoDrop 2000 (Thermo Fisher Scientific, Inc.), the extracted RNA was reversely transcribed into cDNAs using PrimerScript reverse transcriptase kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol, followed by qPCR using SYBR Green kit (Qiagen GmbH) on an ABI7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 10 min initial denaturation at 94˚C, 15 sec denaturation at 94˚C and 30 sec of annealing at 55˚C (40 cycles) and final extension for 1 min at 72˚C. The values were calculated using the 2^-ΔΔCq method (22 (link)) and normalized to U6 or GAPDH expression. The sequences of the primers used were as followed: miR-499a-5p forward, 5'-GCCGAGTTAAGACTTGCAGTGA-3' and reverse, 5'-CTCAACTGGTGTCGTGGA-3'; STAT3 forward, 5'-CATCCTGAAGCTGACCCAGG-3' and reverse, 5'-TCCTCACATGGGGGAGGTAG-3'; GAPDH forward, 5'-AATGGGCAGCCGTTAGGAAA-3' and reverse, 5'-CGCGCCCAATACGACCAAATC-3'; and U6 forward, 5'-AACGCTTCACGAATTTGCGT-3' and reverse, 5'-CTCGCTTCGGCAGCACA-3'.

The quality of the total RNAs was determined at a 260/280 absorbance ratio and through electrophoresis. The RNA sample was stored at −80 °C until further use. After treatment with DNase I, 1 μg of the total RNA was used to synthesise the first strand cDNA with the primerscript reverse transcriptase kit (TaKaRa) according to the manufacturer’s protocol. The SYBR Green RT-PCR assay was conducted using an ABI PRISM 7300 sequence detection system (Applied Biosystems). The thermal profile for SYBR Green RT-PCR was 50 °C for 2 min and 94 °C for 30 s, followed by 40 cycles of 94 °C for 15 s and 58 °C for 40 s. The sequences of the primers are listed in Table S2.

The total RNA was isolated from the mNSCs using the Trizol^® reagent (Invitrogen). The concentrations and quality of RNA were measured using an ND-1000 spectrophotometer (Nanodrop) for further experiments. cDNA was synthesized with a Primer Script Reverse Transcriptase Kit (Takara) according to the manufacturer’s instructions. qPCR was performed using SYBR Premix Ex Taq™ (Takara) on a 7500 Real-Time PCR System. GAPDH was used as the internal reference. Supplementary Figure S1 shows all the primer sequences.