Cfx96 thermal cycler system

Total cellular RNA was extracted using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s protocol. Reverse transcription reactions were performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) with 1 μg of total RNA according to the manufacturer’s protocol. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed with a Bio-Rad CFX96 Thermal Cycler System using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). The results were analyzed by competitive Ct method (Schmittgen and Livak 2008 (link)). The Ct values of specific genes were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) running concurrently to obtain adjusted Ct values (∆Ct). Fold changes were calculated by comparing ∆Ct values from various treatments to control samples. Table S1A shows the primers used for qRT-PCR (Additional file 1).

Total RNA was extracted by TRI Reagent™ solution (Invitrogen, Carlsbad, CA), followed by reverse transcription of 1 μg RNA using Protoscript First Strand cDNA Synthesis Kit (Biolabs, Frankfurt, Germany) as specified by the manufacturer. For quantification of gene expression, qRT-PCR analysis was performed with Maxima SYBR® Green/ROX qPCR Master Mix (Thermo Fisher, Waltham, MA) on a CFX 96 Thermal Cycler system (Bio-Rad, Munich, Germany). Gene expression was calculated by the ΔΔCT method and normalized to β-actin. The following primers were used:
MMP-2, forward 5′- CTCAGATCCGTGGTGAGATCT-3′, reverse 5′- CTTTGGTTCTCCAGCTTCAGG-3′.
MMP-9, forward 5′-ATCCAGTTTGGTGTCGCGGAGC-3′, reverse 5′-GAAGGGGAAGACGCACAGCT-3′.
Beta-Actin, forward 5′-GGCCTCGCTGTCCACCTT-3′, reverse 5′-TGTCACCTTCACCGTTCCAGTTTT-3′.

Total RNA was extracted from MEFs using a NucleoSpin RNA kit (Macherey-Nagel, Bethlehem, PA, 740955) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, 4368814). The RT-qPCR was performed using FastStart SYBR Green Master (Roche, Indianapolis, IN, 04673484001) in a CFX-96 thermal cycler system (Bio-Rad) in accordance with manufacturer’s protocol. Triplicate samples were assessed for each gene of interest, and GAPDH was used as the control gene. Relative expression levels were determined by the 2^−∆∆Ct method. The primers used are given in Table S1.