T100 thermal cycler machine

The extracted DNA was subjected to molecular identification using the SCRABS₆₀₀ marker, Internal Transcribed Spacer region (ITS), and protein-coding gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequencing. The PCR reaction was performed with DreamTaq Green PCR Master Mix (2X) (Thermo Fisher Scientific Inc., United States) by following the manufacturer’s instructions (40 ng of gDNA, 12.5 μL of DreamTaq Green PCR Master Mix (2X), 4 mM MgCl₂, 2 μM of each primer in a final volume of 25 μL). Amplifications were performed on a T100^™ Thermal cycler machine (Bio-RAD, United States).

Total RNA from D341, CHLA-01-MED and CHLA-01R-MED cells were isolated and purified using ISOLATE II RNA Mini Kit (Bioline, London, UK; BIO-52073). The purified RNA was reverse transcribed using SensiFAST™ cDNA Synthesis Kit (Bioline; BIO-65054) and T100™ Thermal Cycler machine (Bio-Rad, Hercules, USA). The target cDNA was amplified using SensiFAST™ Probe No-ROX Kit (BIO-98020; Bioline) with the following primers: CACNA1G forward (5′-GGACTTCTCTTCATGTTGTTG-3′) and reverse (5′-GTCCTTCATAATGCCATTCC-3′); CACNA1H forward (5′-TATCTCGACCTCTTCATCAC-3) and reverse (5′-GACTTGGGTTGGTTATAGTG-3); CACNA1I forward (5′-CTTGGATTGTCATCTTCCAG-3′) and reverse (5′-TGAAGTTGTAGAAGGAGTGAG-3′); CACNA1S forward (5′-AGGAAAACTGTCTTTGGATG-3′) and reverse (5′-TGGATGATTTTGTTCAAGCC-3′); CACNA1C forward (5′-GGAGAGTTTTCCAAAGAGAG-3′) and reverse (5′-TTTGAGATCCTCTTCTAGCTG-3′); CACNA1D forward (5′-AAAATGGGCATCATTCTTCC-3′) and reverse (5′-AGTTTCATAATAGCGGGTTC-3′); CACNA1F forward (5′-CATTTTCACCATCCCAGAAG-3′) and reverse (5′-CTCATCTAGGTAGGAAAGCC-3′); RN18S1 forward (5′-ATCGGGGATTGCAATTATTC-3′) and reverse (5′-CTCACTAAACCATCCAATCG-3′) (Sigma-Aldrich, MO, USA). CFX Connect™ Real-Time PCR Detection System with Starter Package (Bio-Rad) was used to cycle and quantitative targets. The relative target quantity was determined using the comparative CT (ΔΔCT) method by normalising to 18S ribosomal RNA.