Quantitative real-time PCR (qRT-PCR)
This protocol is extracted from research article:
Berberine inhibited metastasis through miR-145/MMP16 axis in vitro
J Ovarian Res, Jan 6, 2021; DOI: 10.1186/s13048-020-00752-2

Total RNA was extracted from cultured cells using Trizol(Invitrogen, Carlsbad, CA, USA)according to the manufacturer’s instructions. According to the OD260/OD280 ratio, the quality of RNA was estimated. The ratio between 1.8 and 2.0 met the experimental requirements. cDNA synthesis was conducted using RevertAid first strand cDNA synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using a SYBR Premix Ex Taq™ II kit (Takara, Dalian, China). miR-145 were normalized to U6, while MMP16 were normalized to the gene β-actin. Relative gene expression was calculated automatically using 2−ΔΔCt. The following primer sequences of human genes were used:

MMP16 forward: 5′- GGACAGAAATGGCAGCACAAGC-3′;

MMP16 reverse: 5′- CATCAAAGGCACGGCGAATAGC-3′;

β-actin forward: 5’-TCCCTGGAGAAGAGCTACGA-3’;

β-actin reverse: 5’-AGCACTGTGTTGGCGTACAG-3’.

Note: The content above has been extracted from a research article, so it may not display correctly.



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