Primescript rt reagent

At 24 h post-transfection, cells were treated with TRIzol (Takara Bio, Inc., Otsu, Japan) to extract total RNA, which was reverse transcribed to first strand cDNA using Primescript RT Reagent (Takara Bio, Inc.). The expression levels of miR-675-3p (sense 5′-CGGAGAGGGCCCACAGTG-3′; anti-sense was an universal primer which was obtained from GeneCopoeia, USA), MMP2 (sense 5′-GCTGACGGTAAGGACGGACTC-3′; anti-sense 5′-CGTTGCCATTGAACAAGAAGG-3′), MMP9 (sense 5′-TGTGCTACAGGGAGAGATAAGA-3′; anti-sense 5′-GTGGGTGGAGCAGAGTAAATAA-3′) and E-cadherin (sense 5′-CTCAGTTGGAACAGGGTGAATA-3′; anti-sense 5′-GTGCAGGACACTCAAATCAAAG-3′) were examined using RT-qPCR in a Step one plus system (Roche Molecular Diagnostics, Pleasanton, CA, USA) using 2^−ΔΔCq analysis. In this system, all reactions were performed in SybrGreen (ChamQ^™ SYBR qPCR Master Mix; Vazyme, Piscataway, NJ, USA), in a volume of 20 ul containing 2 ul cDNA, according to the manufacturer's protocol. All reactions were performed in triplicate. The thermal cycling conditions included a step of 5 min at 95°C followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. U6 was used as internal control. Final amounts of target in cells were determined as follows: Target amount=2^−ΔΔCq, where ΔΔCq=[Cq (target)-Cq (U6)]sample-[Cq (target)-Cq (U6)]internal standard. Final amounts of target in tissues were determined as target amount=2^−ΔΔCq (14 (link)).