Miniopticon real time pcr detection system

SW1353 cells (4 × 10⁵) were seeded in a 6-cm dish and treated with MIA and/or zinc for 24 h, and then the total RNA was extracted by using REzol reagent (Protech, Taipei, Taiwan) according to the manufacturer’s instructions, as previously described [19 (link)]. Each experiment was carried out in triplicate. The complementary DNA (cDNA) was synthesized from random primed reverse transcription from 2 μg of total RNA using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. Real-time PCR was performed on a MiniOpticon^TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iQ^TM SYBR^® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) as previously described [20 (link)], was used to confirm the results of real-time PCR. The mRNAs encoding GCLC, GCLM, IL-10, and IL-1β were measured using real-time PCR, with RPS18 mRNA as the housekeeping gene. The primers and amplified products of each gene used in the present study are shown in Table 1. The cycle threshold (C_t) value of the target gene was normalized to RPS18. The data were calculated and expressed as 2^−ΔΔCt [21 (link)] using MJ Opticon Monitor Analysis software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA).

Total RNA was reverse transcribed into complementary DNA using a Transcriptor Reverse Transcriptase kit (Roche Applied Science, Indianapolis, IN, USA). Quantitative reverse transcription-PCR was performed using a MiniopticonTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Inc., Boston, MA, USA). The internal control gene was 18S rRNA. The primers are as follows: glucose transporter 4 (glut4) forward: 5′-ACATACCTGACAGGGCAAGG′-3′ and reverse: 5′-CGCCCTTAGTTGGTCAGAAG′-3′; 18S rRNA (18S) forward: 5′-ACGATGCCGACTGGCGATGC-3′ and reverse: 5′- TCCTGGTGGTGCCCTTCCGT-3′. mRNA expression of each gene was normalized to the 18S gene in the same sample. Threshold cycle (Ct) values were obtained and the relative gene expression was calculated using the formula 2^−ΔΔCt.

Quantitative polymerase chain reaction (qRT-PCR) was performed in a final volume of 20 µl consisting of 1 µl of the cDNA, 10 µl of SensiFAST SYBR No-ROX kit (Roche) and 15 µM of the gene-specific primers pairs as follows: SbPPC3 (forward 5′–TGTTGAACAGTTTCTGGAACCTCTT-3′, reverse 5′-GCTTCA CAAGGGCAAGCCCAAAG-3′), SbPPC2 (forward 5′-CCGCCT CGCAACACCTGAAACA-3′, reverse 5′-ACCGGGAGGTGGAA CCGTGT-3′), SbPPC4 (forward 5′-TGAGCTTCGGGCACAAGC AGATG-3′, reverse 5′-GCTCCAAAGGCTCTAAGAACTGCT C-3′), and 18 S rRNA (forward 5′-GGGGAAACTTACCA GGTCCA-3′, reverse 5′-GGATGGCTCCGCATAGCTA-3′). PCR was conducted on the MiniOpticon^TM Real-Time PCR Detection System (Biorad), and the threshold cycles (Ct) were determined using Opticon Monitor^TM analysis software for all treatments. To normalize the obtained values, 18S ribosomal RNA was used as internal control in each sample. This gene displays a steady RNA level across the experimental conditions.

Chromatin immunoprecipitation (ChIP) assay was performed following the protocol described previously (Carey et al., 2009). Briefly, cells (5 × 10⁷) were seeded in 100‐mm dishes overnight before being fixed with 1% formaldehyde for 10 min, followed by a 5‐min treatment with 1.25 m glycine to quench the reaction. The cell pellet was then resuspended in a cell lysis buffer [5 mm PIPES, pH 8.0; 85 mm KCl; 0.5% nonidet P‐40 (NP‐40)] and incubated on ice for 10 min before the nuclei were collected by centrifugation at 157 g for 10 min at 4 °C. Subsequently, the nuclei were resuspended in 1 mL of nuclei lysis buffer (50 mm Tris/Cl, pH 8.0; 10 mm EDTA; 1% SDS) supplemented with a protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) and then incubated on ice for 10 min. Sonication was then applied to shear DNA into 500‐bp fragments, and precleared lysates were subjected to overnight immunoprecipitation with 2 µg·mL⁻¹ of rabbit anti‐GATA6 antibody or normal rabbit IgG (negative control). After phenol/chloroform extraction, DNA samples were recovered by isopropanol precipitation and aliquots from the resuspended DNA pellets were subjected to a SYBR green‐based quantitative PCR assay using primers (Table S1) with a MiniOpticonTM Real‐Time PCR Detection System (Bio‐Rad).

Total RNA was extracted from cells with REzol reagent (Protech, Taipei, Taiwan) according to the manufacturer’s instructions, as described previously [38 (link)]. The complementary DNA (cDNA) was synthesized from random primed reverse transcription from 2 μg of total RNA using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer’s directions. Real-time PCR, performed on a MiniOpticonTM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iQTM SYBR^® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) according to a published procedure [39 (link)], was used to confirm results of real-time PCR. mRNA coding for MMP-1, MMP-3, MMP-9, MMP-13, IL-1β, IL-6, IL-17A, and TNF-α were measured by real-time PCR, with β-actin mRNA being amplified as a housekeeping gene. Primer sequences of targets are listed in Table 1. The cycle threshold (C_t) value of the target gene was corrected by the β-actin. Data were calculated and expressed as ΔΔC_t [40 (link)] by using MJ Opticon Monitor Analysis software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA).

The RNA Seq data were validated by quantitative real-time PCR (qRT-PCR). Synthesis of cDNA was carried out using iScript^TM Reverse Transcription (Bio-Rad Laboratories, Inc. Hercules, CA, USA). qRT-PCR was performed on a MiniOpticon^TM Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with iQTM SYBR^® Green Supermix kit (Bio-Rad Laboratories, Hercules, CA, USA). Gene rtcR, encoding for transcriptional regulator protein RtcR, was selected as an internal reference gene. The expression of rtcR in the wild type and its isogenic yccT mutant was not affected by H₂O₂ treatment or yccT mutation. Primers of seven genes (ego, pduA, ydeZ, ydeV, yneC, rplP and rplV) were designed to generate internal fragments ranging from 91 bp to 129 bp in size (Table S1).

RNA was extracted from samples in different groups (n = 5 per group) using the DirectzolTM RNA MiniPrep kit (Zymo Research, USA) and reverse transcribed into cDNA using the qScriptTM cDNA Synthesis kit (Quanta Biosciences, USA) and a MiniOpticon real-time PCR detection system (BioRad, USA), according to the manufacturer's instructions. Polymerase chain reaction (PCR) was conducted using a PerfeCta SYBR Green FastMix kit (Quanta Biosciences, USA) according to the manufacturer's instructions. The expression genes were analyzed using an ABI 7300 real-time PCR System (Thermo Fisher, USA). The expression of proteoglycan 4 (PRG4), cartilage intermediate layer protein 1 (CILP), collagen type II (COL2A1), collagen type I (COL1A1), and SOX-9 (SOX9) was normalized to the expression level of the housekeeping gene for β-actin (ACTB). The primer sequences for PRG4, CILP, COL2A1, COL1A1, SOX9, and ACTB are listed in Supplementary Table 1.