Toprealtm qpcr 2x premix

Total RNA was extracted using a Hybrid-R RNA extraction kit (GeneAll Biotechnology, Seoul, South Korea). cDNA was synthesized by M-MLV cDNA Synthesis kit (Enzynomics, Daejeon, South Korea) according to the supplier’s instructions. Quantitative real-time PCR was performed using TOPrealTM qPCR 2X PreMIX (Enzynomics) on a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA). Primers used were 5’-AGGGCATCATCAATTTCGAG-3’ (sense) and 5’-TGCCTCTCTTCATCCTTTGG-3’ (antisense) for human SOD1 (NCBI gene ID: 6647); 5’-GTGTGATGGTCCTTCCAACC-3’ (sense) and 5’-CTGACATCCTCTGGCTCACA-3’ (antisense) for human Prdx6 (NCBI gene ID: 9588); 5’-CAGTCTCAAGTATGTCCGT-3’ (sense) and 5’-AGGCTCAATGTTGATGGT-3’ (antisense) for human Gpx2 (NCBI gene ID: 2877); 5’-TTGGCTACCTTGCAGTTCGT-3’ (sense) and 5’-ATGTGAACCATCGCAGGCA-3’ (antisense) for human CISD2 (NCBI gene ID: 493856); 5’-CATGTACGTTGCTATCCAGGC-3’ (sense) and 5’-CTCCTTAATGTCACGCACGAT-3’ (antisense) for human β-actin (NCBI gene ID: 60). Ratio of target gene fold-change was normalized to human β-actin expression using comparative CT (2-ΔΔCt) method.

Total RNA was extracted from samples using TRIzol reagent according to the appropriate protocols, and the amount of RNA was measured using Nanodrop (Thermo Scientific, Waltham, MA, USA). Then, cDNA was synthesized from 1 μg extracted total RNA using SuperScriptTM III Reverse Transcriptase (Invitrogen, Waltham, MA, USA, 18080-044), Oligo(dT)12-18 Primer (Invitrogen, 18418-012), and 10 mM dNTP Mix (Invitrogen, 18427-013). Real-time polymerase chain reaction (real-time PCR) was carried out using TOPrealTM qPCR 2X PreMIX (Enzynomics, Daejeon, Korea, RT500M), and the results were analyzed using a Roche LightCycler 5480 (Roche). The primers used for real-time RT-PCR are listed in Table S1.

The total RNA content was extracted from three samples per test condition using the TRIzol reagent (Bioneer, Daejon, South Korea). The Nanodrop 2000 was used to examine the RNA (Thermo Fisher Scientific, MA, USA). To reverse-transcribe RNA into cDNA, the ReverTra Ace™ qPCR RT master mix (Toyobo, Osaka, Japan) was used. TOPreal^TM qPCR 2X premix (Enzynomics, Daejon, Republic of Korea) and CFX96 Dx real-time PCR detection equipment (Biorad, CA, USA) were utilized to perform qPCR. The Ct values of all samples were obtained in triplicates. Average Ct values were used to calculate the gene expression fold change. As a housekeeping gene, β-actin was employed. For the expression analysis, the 2^−ΔΔCT approach was used. The oligoprimers utilized for qPCR are listed in Table S2.

Total RNA isolation from cells or tissues was performed by using TRIzol reagent (15596026, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Isolated RNA was subsequently reverse-transcribed to complementary DNA by using the TOPscript^TM RT DryMIX kit (RT200, Enzynomics, Daejeon, Korea). Analysis of quantitative real-time PCR was conducted with an ABI 7300 (Applied Biosystems, Foster City, CA, USA) by using TOPreal^TM qPCR 2X PreMIX (RT501M, Enzynomics, Daejeon, Korea). The relative mRNA expression of each target was normalized to cyclophilin A or Gapdh. Mouse primer sequences are available on request (CycloA, Gapdh, Fbp1, Fbp2, Pygm, G6pi, Pfk1, Pgm2, Gys1, PGC-1α, Tfam, Nrf-1, Cox5b, Atp5b, Ndufv5).

Total RNA was isolated from various MSCs cultures using the Direct-zol™ RNA MiniPrep (Zymo Research Corporation, Irvine, CA, U.S.A.) according to the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and reverse transcription reactions were performed using 0.2 μg of total RNA with a TOPscriptTM cDNA synthesis kit (enzynomics, Daejeon, Korea). The real-time PCRs for beta-actin, collagen II, and aggrecan were performed using the TOPrealTM qPCR 2X Pre MIX (enzynomics). Primer sequences are listed in Table 1. Real-time PCRs were performed using a StepOnePlus™ instrument (Applied Biosystems, Grand Island, NY, USA) at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 10 s, extension at 60 °C for 15 s, and annealing at 72 °C for 15 s. Gene expression levels were normalized to that of beta-actin and relative gene expression was calculated using the ddCT method.

CREC was grown under the influence of fingolimod and doripenem in TSB until it reached the exponential phase. Cells were collected by centrifuging 2 mL of bacterial suspensions from each sample at 25,000 g for 10 min at 4 °C. For RNA isolation and purification, a NucleoSpin RNA Mini Kit (Macherey-Nagel, Düren, Germany) was used, following the manufacturer’s instructions. The BioDrop μLITE (BioDrop Ltd., Cambridge, UK) was used to measure the quality and concentration of the isolated RNA samples. cDNA was synthesized using ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan) according to the manufacturer’s protocols. TOPreal^TM qPCR 2X PreMIX (Enzynomics, Daejeon, Korea) was used to amplify cDNA in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The qPCR cycling conditions were as follows: initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 s, primer annealing for 15 s, extension at 72 °C for 30 s, and finally, a melting-curve step (95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s). Table 1 shows the primers and annealing temperature of the carbapenemase- and virulence-related genes. The mRNA expression levels of the target genes were normalized against those of 16S rRNA, a housekeeping gene, and the 2^−∆∆CT formula was used.

Primers were chosen with Primer3 design program [18 (link)]. The primer sets used in this study are shown in Table 1. Total RNA was isolated from cells with TRIzol reagent according to the manufacturer's protocol. First-strand cDNA was synthesized with 0.5 μg of total RNA, 1 μM oligo-dT18 primer, and the M-MLV cDNA synthesis kit (Enzynomics, KR) according to the manufacturer's protocol. SYBR green-based QPCR was performed with the Stratagene Mx3000P real-time PCR system and TOPrealTM qPCR 2X PreMIX (Enzynomics, KR), with the first-strand cDNA diluted 1 : 10 and 20 pmol of primers according to the manufacturer's protocol. The polymerase was activated at 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 s (denaturation), 60°C for 30 s (annealing), and 72°C for 30 s (extension). This was followed by the generation of PCR-product temperature-dissociation curves (also called melting curves) at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s. All reactions were run in triplicate, and data were analyzed by the 2^−ΔΔCT method [19 (link)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard.