Quantstudio 6 and 7 flex real time pcr system software v1

RNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. For gene expression analysis, cDNA was generated from 1μg of RNA with the iScript cDNA synthesis kit (Bio-Rad) as per the manufacturer’s protocol. Using the QuantiTect SYBR Green PCR kit, 100ng of cDNA was amplified according to the manufacturer’s instructions with primers targeting BUB1B (catalog no. QT00008701), MAD2 (catalog no. QT00094955), TTK (catalog no. QT00035168), KIF18A (catalog no. QT00042455), or GAPDH (catalog no. QT00273322) as an endogenous control (Quantitect Primer Assay, Qiagen). Data analysis was performed with the QuantStudio 6 and 7 Flex Real-Time PCR System Software v1.0 (Applied Biosystems, Life Technologies) using the ΔΔCt method.

Total RNA from BV-2 cells exposed to FRs for 18 hr was isolated using TRIzol® Reagent (Invitrogen, Carlsbad, CA), quality assessed by NanoDrop (Thermos Scientific, Wilmington, DE), and complementary deoxyribonucleic acid (cDNA) synthesized from 2.5 μg total RNA using SuperScript™ II reverse transcriptase with random hexamers (Invitrogen). qPCR was performed in duplicate using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems; Foster City, CA) with TaqMan® detection of reaction products. A 2.5 μL cDNA template was added with 1X Power TaqMan® Universal PCR Master Mix (ThermoFisher Waltham, MA). The 20 μL PCR reaction mixtures were held at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 1 min at 60°C. Amplification curves were generated with QuantStudio™ 6 and 7 Flex Real-Time PCR System Software v1.0 (Applied Biosystems). Data was obtained from samples that met inclusion criteria of transcript detection at <32 cycles. mRNA levels were calculated relative to Gapdh. and compared to vehicle control using the 2^-ΔΔCT method.