Chondrocytes (1 × 105 in each well) cultured in a six-well plate were washed with cold PBS and incubated with TRIzol reagent to extract total RNA. cDNA was synthesized using 1 mg total RNA with a cDNA synthesis kit (MBI Fermantas, Germany). The primers of MMP3, -9 and-13, ADAMTS4 and-5, CCL-2 and-5, and CXCL1 are shown in Table 1 . tive mRNA levels of those genes were calculated using the 2−ΔΔCt method. Twenty microliters of reaction volume was prepared before amplification, including 2 μL of twofold diluted cDNA, 10 μL 2 × SYBR Premix Ex Taq mixture (Takara, Japan), 0.2 μM each primer, and sterile distilled water. The amplification reaction was performed on a Mastercycler® ep realplex platform (Eppendorf, Hamburg, Germany). After amplification, the cycle threshold (Ct) values were obtained and normalized to the value of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. The relative mRNA levels of those genes were calculated using the 2−ΔΔCt method.
Mastercycler ep realplex platform
Manufactured by Eppendorf
Sourced in Germany
The Mastercycler Ep-Realplex platform is a real-time PCR system designed for high-throughput nucleic acid quantification and gene expression analysis. It features a compact, modular design and supports multiple detection formats, including SYBR Green, hydrolysis probes, and melt curve analysis.
Automatically generated - may contain errors
Lab products found in correlation
Power sybr green reagent, by Thermo Fisher Scientific (3 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq mixture, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Quantstudio real time pcr system, by Thermo Fisher Scientific (1 mentions)
4 protocols using mastercycler ep realplex platform
1
Chondrocyte Gene Expression Analysis
2
RNA Extraction and Real-Time qPCR Analysis
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Total RNA from cells and digested tissue pellets was extracted using the TRIzol™ Reagent (Thermo Fisher Scientific) and isolated according to the manufacturer’s instructions. Target genes were evaluated by quantitative RT-qPCR on a Mastercycler Ep-Realplex platform (Eppendorf, Hamburg, Germany), using Power SYBR Green reagents (Applied Biosystems, Foster City, CA, USA). Cycle conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Quantified gene expression values were normalized against those of GUSB, U6, or GAPDH (Table S1 ); when indicated, WPRE sequence [42 (link)] was evaluated as a subrogated indicator for vector presence or expression (Table S1 ).
To detect and quantify the specific miRNA, TaqMan™ MicroRNA Assays (Thermo Fisher) were used according to the manufacturer’s instructions (Table S1 ). The real-time PCR amplification was carried out in a QuantStudio real-time PCR system (Thermo Fisher Scientific) under the following conditions: 95 °C for 10 min, then 95 °C for 15 s, and 60 °C for 60 s for up to 40 cycles.
To detect and quantify the specific miRNA, TaqMan™ MicroRNA Assays (Thermo Fisher) were used according to the manufacturer’s instructions (
3
RT-qPCR Analysis of Gene Expression
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Total mRNA was isolated as described33 (link). cDNA first strands were synthesized from total RNA (1 μg) with the SuperScript III First-Strand Synthesis System (Invitrogen). Genes of interest (see Supplementary "Methods " section online) were evaluated by quantitative RT-qPCR in a Mastercycler Ep-Realplex platform (Eppendorf, Hamburg, Germany), using Power SYBR Green reagents (Applied Biosystems, Foster City, CA). Cycle conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Quantified gene expression values were normalized against those of GUSB or GAPDH. Supplementary Methods Table 1 section online includes the list of all primer sequences used.
4
Quantifying Gene Expression and Cell Viability
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Total mRNA was isolated as described [19] . cDNA rst strands were synthesized from total RNA (1 mg) with the SuperScript III First-Strand Synthesis System (Invitrogen). Genes of interest (see Supplementary Methods section online) were evaluated by quantitative RT-qPCR in a Mastercycler Ep-Realplex platform (Eppendorf, Hamburg, Germany), using Power SYBR Green reagents (Applied Biosystems, Foster City, CA). Cycle conditions were 95°C for 10 min, followed by 40 cycles of 95ºC for 15 s and 60°C for 1 min.
Quanti ed gene expression values were normalized against those of GUSB or GAPDH.
Biotech, Birmingham, AL). Labeled cells were washed with PBS/0.01% BSA and resuspended in 390 mL of binding buffer. Propidium iodide (50 mg/ml, Beckman Couler, Nyon, Switzerland) was added (1:40 dilution) for dual-staining and cells were analyzed by ow cytometry. DAPI and AnexinV/PI positive-cells were quanti ed on a FACS Canto 3L ow cytometer (BD Biosciences). When indicated, necrosis of hCPC was induced by a short heat treatment (10 min, 60ºC) of attached monolayers.
Quanti ed gene expression values were normalized against those of GUSB or GAPDH.
Biotech, Birmingham, AL). Labeled cells were washed with PBS/0.01% BSA and resuspended in 390 mL of binding buffer. Propidium iodide (50 mg/ml, Beckman Couler, Nyon, Switzerland) was added (1:40 dilution) for dual-staining and cells were analyzed by ow cytometry. DAPI and AnexinV/PI positive-cells were quanti ed on a FACS Canto 3L ow cytometer (BD Biosciences). When indicated, necrosis of hCPC was induced by a short heat treatment (10 min, 60ºC) of attached monolayers.
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