Applied biosystems high capacity reverse transcription kit

RNA was prepared using the Qiagen RNeasy Plus Kit (74136, Qiagen) according to the manufacturer’s instructions and reverse-transcribed to cDNA using the Applied Biosystems High-Capacity Reverse Transcription Kit (43-688-13, Thermo Fisher). Relative expression was calculated as previously described^{16 (link)} on an Applied Biosystems Quantstudio 6 by the 2^−ΔΔCt method^{56 (link)} using β-actin (ACTB) as an internal control. The following primers were used:
ACTB forward: 5′-GGACTTCGAGCAAGAGATGG-3′
ACTB reverse: 5′-AGGAAGGAAGGCTGGAAGAG-3′
HEYL forward: 5′-CTCCAAAGAATCTGTGATGCCAC-3′
HEYL reverse: 5′-CCAGGGACAATGAAAGCAAGTTC-3′
HEY1 forward: 5′-CCGCTGATAGGTTAGGTCTCATTTG-3′
HEY1 reverse: 5′-TCTTTGTGTTGCTGGGGCTG-3′
HES1 forward: 5′-ACGTGCGAGGGCGTTAATAC-3′
HES1 reverse: 5′-ATTGATCTGGGTCATGCAGTTG-3′
HMGA1 forward: 5′-GAAAAGGACGGCACTGAGAA-3′
HMGA1 reverse: 5′-TGGTTTCCTTCCTGGAGTTG-3′
HMGA2 forward: 5′-AGCGCCTCAGAAGAGAGGA-3′
HMGA2 reverse: 5′-AACTTGTTGTGGCCATTTCC-3′

RNA was prepared using the Qiagen RNeasy Plus Kit (74136, Qiagen) according to the manufacturer’s instructions and reverse-transcribed to cDNA using the Applied Biosystems High-Capacity Reverse Transcription Kit (43-688-13, Thermo Fisher). Relative expression was calculated as previously described^{31 (link)} on an Applied Biosystems Quantstudio 6 by the 2^−ΔΔCt method using β-actin (ACTB) as an internal control. The primers used are mentioned in Supplementary Data 7.

The K7M2 cell line was homogenized in 1 mL of TRIzol-reagent by vortexing, and its RNA contents were isolated using Direct-zol™ RNA MicroPrep (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. The amount and purity of RNA were determined using a Jenway™ Genova Nano Micro-volume Spectrophotometer (Fisher Scientific, Loughborough, UK). Samples were then treated with 1U DNase I enzyme to eliminate any contaminating genomic DNA. cDNA was synthesized from 500 ng of RNA using an Applied Biosystems™ High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA).
PCR was performed using a QuantStudio™ 5 system (Life Technologies Magyarország Ltd., Budapest, Hungary) in a 96-well block, using Gapdh as a reference gene, with a reaction volume of 10 µL, containing 1× SensiFAST™ Probe Lo-ROX mix (Meridiane Bioscience, Memphis, TN, USA), 400 nM of probe primer mix (forward and reverse), and 20 ng cDNA. FAM-conjugated TaqMan™ Gene Expression Assays (Thermo Scientific, Waltham, MA, USA) were used to amplify the target loci—Gapdh: Mm99999915_g1, Trpa1: Mm01227437_m1, and Trpv1: Mm01246302_m1. The K7M2 PCR products were electrophoresed on a 2% agarose gel containing 0.01% ethidium bromide at 70 V for 40 min and visualized using a Molecular Imager BioRad Gel Doc XR+ (BioRad Laboratories, Hercules, CA, USA) with Image Lab 6.0.1 build 34 software.