Cultured cell total rna purification mini kit

Total RNA was extracted from the cell lines using a Cultured Cell Total RNA Purification Mini Kit (FAVORGEN, Ping Tung, Taiwan) followed by reverse transcription using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) according to manufacturer’s instructions. cDNA was amplified for 40 cycles in a LightCycler 480 instrument (Roche, Basel, Switzerland) using LightCycler 480 SYBR Green I Master reagent (Roche). The primer sets used for qPCR are: for Par3, 5′-CGCTTGGAACATGGAGATGG-3 and 5′-ATCTCTGGGCTCTGGGTACC-3, for GAPDH, 5′-GAAAGGTGAAGGTCGGAGTC-3 and 5′-GAAGATGGTGATGGGATTTC-3. mRNA levels of each gene were normalized to GAPDH mRNA as an internal standard. Expression levels were calculated by the comparative Ct method using GAPDH as the endogenous reference gene.

Total RNA was extracted using the Cultured Cell Total RNA Purification Mini Kit (Favorgen, Germany). After extraction, total RNA concentration and purity were evaluated using an Agilent 2100 Bio-analyzer system and an Agilent Small RNA analysis kit (Agilent technologies, Waldbronn, Germany). RNA (1 μg per sample) was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit from Applied Biosystems (Warrington, USA) at 37 °C for 2 h.