Mirna purification kit

TRIzol^® (Invitrogen; Thermo Fisher Scientific Inc.) reagent was added to patient tissues and cells lines (MCF-10A, SK-BR-3, MCF-7, T-47D and MDA-MB-231) according to the manufacturer's instructions. miRNA was purified from tissues and cells using a miRNA Purification kit (CoWin Biosciences). The extracted total RNA was then reverse transcribed into cDNA using the miRNA cDNA Synthesis kit (CoWin Biosciences) according to the manufacturer's protocol. qPCR was performed on a 7300 real-time PCR system using a SYBR Green miRNA qPCR Assay kit (CoWin Biosciences). The thermocycling condition used were as follows: 95°C for 10 min followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 70°C for 30 sec. The relative expression of miR-768-3p was calculated by the 2^−ΔΔCq method (16 (link)), and U6 was used as the reference gene for mRNA quantification. The following primer sequences were used: miR-768-3p forward 5′-GCCGAGUCACAAUGCUGACACUCA-3′ and reverse 5′-CTCGTTCGGCAGCACA-3′; and U6 forward 5′-AACGCTTCACGAATTTGCGT-3′ and reverse 5′-CTCGTTCGGCAGCACA-3′. Each sample was tested in triplicate.

After the transfection for 24 hours, total RNA was extracted using TRIzol reagent (Invitrogen, USA) and reverse transcribed to cDNA using a HiFiScript cDNA Synthesis Kit (CoWin Biotech Co. Ltd., China). MiRNA was extracted using an miRNA Purification Kit (CoWin Biotech Co. Ltd., China) and reverse transcribed to cDNA using an miRNA cDNA Synthesis Kit (CoWin Biotech Co. Ltd., China). UltraSYBR Mixture and miRNA qPCR Assay Kit (CoWin Biotech Co. Ltd., China) were used for qPCR according to the manufacturer’s protocol. The following primers were used in this study, miR-218-5p (5ʹ-TTGTGCTTGATCTAACCATGT-3ʹ), NACC1-Forward (5ʹ-GCCTGTACTGTGACGTGTCA-3ʹ) and NACC1-Reverse (5ʹ-CGTGGAGAACTTGGCCATCT-3ʹ). The relative expressions of genes were calculated using the 2^−ΔΔCt method.