Iscript reverse transcription supermix for rt qpcr

Total RNA was synthesized using the RNA extraction kit (Qiagen, Germany) following the manufacturer’s protocol. The RNA samples were stored at −80°C until use. For quantitative gene expression analyses, high-capacity complementary DNA (cDNA) was generated from 2 μg of RNA using the iScriptTMReverse Transcription Supermix for RT-qPCR (Bio-Rad, Foster City, CA, United States). Then, cDNA was diluted to a ratio of 1:10 with SsoFast^TM EvaGreen^® Supermix with Low ROX qRT-PCR (Bio-Rad, Foster City, CA, United States), mixed with forward and reverse primers of specific genes (Table 1). Real-time PCR was first performed by holding the sample at 95°C for 30 s. Then, the PCR sample was set for denaturing at 95°C for 5 s and to annealing/extension at 60°C for 30 s. After 44 cycles repeat, the sample was then heated up to 95°C for melt curve analysis. Eventually, EvaGreen fluorescence was detected by the CFX96 Touch Real-time PCR detection system (Bio-Rad, Foster City, CA, United States), and Cq values were obtained. All targeted genes were measured in triplicate, and three independent biological triplicates were detected in each condition. The Cq values were then calculated via 2-(ΔΔCq) equation representing relative fold change in the expression of each gene. Relative mRNA expression levels were normalized using reference internal control gene, act-1.

Cells were cultured and treated in 8-well plates (SCC-1, SCC-9, CAL-27, SCC-6, SCC-47, SCC-104, SCC-90 and SCC-152) and washed with 3 × 2 ml PBS, with the exception of UM-SCC-6 which were washed only 1 × 2 ml, cells were scraped, and transferred in 300 μL RNA Later (cat: AM7021, ThermoFisher Scientific) to 1.7 mL centrifuge tubes. The tubes were vortexed, and 20 μL of sample was transferred to a new centrifuge tube. RNA purification was performed using the RNeasy Plus Mini Kit supplemented with gDNA Eliminator mini Spin Columns (Cat#: 74134 Qiagen) and QIAshredder (Cat#: 79656 Qiagen) per manufacturers guidelines. RNA was used to synthesize cDNA using the iScriptTM Reverse Transcription Supermix for RT-qPCR (Cat#: 1708841 BIO-RAD) as per the manufacturer’s guidelines. Following cDNA synthesis, samples were diluted with Nuclease-free H2O to 1 ng/μl and either stored at -20 °C or used directly for droplet digital PCR (ddPCR).

RT-PCR primers for detection of GAS6 mRNAs are as below:
hGAS6-RT-PCR-F1: 5′-GCAAAACCTGCCTGACCAGT-3′
hGAS6-RT-PCR-R1: 5′-TTCGTTGACATCTTTGTCGCA-3′
β-actin-F: 5′-CCTGGCACCCAGCACAAT-3′
β-actin-R: 5′-GCCGATCCACACGGAGTA-3′
RT-PCR experiments were performed as below:
Total cellular RNA was extracted using RNeasy mini kit (QIAGEN) according to the manufacturer’s protocol and quantified by a spectrophotometer (Nanodrop One C). cDNA was synthesized using iScript^TM Reverse Transcription Supermix for RT-qPCR (Bio-Rad Cat#1708841). cDNA templates and iTaq^TM universal SYBR Green Supermix (Bio-Rad Cat#1725122) were mixed together and the RT-PCR reaction was performed on the ViiA^TM 7 Real-Time PCR system. The expression of GAS6 and PROS mRNAs was normalized to the expression of β-actin.