Total RNA from DC and CD4+ T cells was extracted using an RNApure Tissue Kit (Millipore, Darmstadt, Germany) and reverse-transcribed to cDNA using the HiFiScript 1st Strand cDNA Synthesis Kit (CWBIO, Beijing, China). Real-time PCR was performed using TransStart Green qPCR SuperMix (Transgen, Beijing, China) and tested using the DA7600 real-time nucleic acid amplification fluorescence detection system (Bio-Rad, CA). Primers synthesized by the Beijing Genomics Institute are listed in Supplementary Table 1 . Data are expressed using the CT method and normalized against housekeeping gene (GAPDH) levels.
Da7600 real time nucleic acid amplification fluorescence detection system
Manufactured by Bio-Rad
Sourced in United States
The DA7600 Real-time Nucleic Acid Amplification Fluorescence Detection System is a laboratory instrument designed for real-time monitoring and analysis of nucleic acid amplification processes. The system utilizes fluorescence detection to track the amplification of target nucleic acid sequences in real-time. The core function of the DA7600 is to provide a platform for sensitive and precise measurement of nucleic acid amplification, allowing researchers to quantify and characterize target molecules during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Transstart green qpcr supermix, by Transgene (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Eastep total rna extraction kit, by Promega (2 mentions)
Hifiscript 1st strand cdna synthesis kit, by CWBIO (1 mentions)
Transscript first stand cdna synthesis kit, by Transgene (1 mentions)
Cytoplasmic and nuclear rna purification kit, by Norgen Biotek (1 mentions)
Sybr green pcr master mix, by Qiagen (1 mentions)
Goscript reverse transcriptase, by Promega (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Easyscript reverse transcriptase, by Transgene (1 mentions)
6 protocols using da7600 real time nucleic acid amplification fluorescence detection system
1
Quantifying gene expression in immune cells
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from the cultured cells using an Eastep Total RNA Extraction kit (Promega) according to the manufacturer’s protocol. Then, total RNA was reverse transcribed using a TransScript First-Stand cDNA Synthesis kit (TransGen Biotech). RT-qPCR was subsequently performed with TransStart Green qPCR SuperMix (TransGen Biotech), and products were detected with a DA7600 Real-time Nucleic Acid Amplification Fluorescence Detection System (Bio-Rad, Hercules, CA, USA). We quantified the transcripts of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal mRNA quantity control. All primers for cDNA amplification of various target genes were designed and optimized using Oligo 7.0 software (Molecular Biology Insights, West Cascade, USA) and synthesised by Sangon Biotech (Shanghai, China). The relative expression levels of the target genes were calculated by normalizing the cycle threshold (Ct) values of the target gene to the Ct values of GAPDH (ΔCt) and determined as 2-ΔCt. The RT-qPCR products were subjected to electrophoresis.
3
mRNA Extraction and Quantification
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Total mRNA was obtained from tissues and cells with the help of Total RNA Extraction TRIzol reagent (Baiaolaibo Co., Ltd., Beijing, P.R. China). Cytoplasmic and nuclear RNAs were extracted from SKOV3 and OVCAR3 cells using the Cytoplasmic and Nuclear RNA Purification kit (Norgen Biotek, Ontario, Canada), according to the manufacturer’s protocols. Then a total of 1 mg of mRNA was reversely transcribed (RT) to the first-strand cDNA using the First-Strand cDNA Synthesis kit for Real-Time PCR (Thermo Fisher Scientific), followed by qPCR procures using TransStart Green qPCR SuperMix (Thermo Fisher Scientific) on DA7600 Real-time Nucleic Acid Amplification Fluorescence Detection System (BioRad, Berkeley, CA, USA). Melting curve was conducted to analyze the reaction specificity. GAPDH served as an internal reference. Primers were obtained from Sangon Biotech (Shanghai, P.R. China) and are listed in Table 1 .
4
Quantitative Analysis of Stem Cell Markers in Ovarian Cancer Cell Lines
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Total RNA from SKOV3 or A2780 cells was obtained using TRIzol reagent (Thermo Fisher Scientific) when the cells reached confluence of 90%, referring to the manufacturer’s instructions. The ThermoScript reverse-transcription PCR system (Promega Corporation, Fitchburg, WI, USA) was used to reverse RNA to cDNA, according to the manufacturer’s protocols. Quantitative RT-PCR was carried out by SYBR Green PCR master mix (Qiagen NV, Venlo, the Netherlands) in a 20 µL reaction system on a DA7600 Real-time Nucleic Acid Amplification Fluorescence Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The primers used were as follows: OCT4, 5′-ATGTGGTCCGAGTGTGGTTC-3′ (forward) and 5′-GAGACAGGGGGAAAGGCTTC-3′ (reverse); GAPDH, 5′-ATCATCCCTGCCTCTACTGG-3′ (forward) and GAPDH, 5′-GTCAGGTCCACCACTGACAC-3′ (reverse).
5
Quantitative RT-PCR Analysis of mRNA
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Total RNA was obtained using an Eastep total RNA extraction kit (Promega, Wisconsin, WI, USA). Total RNA from each sample was then subjected to reverse transcription using GoScriptTM reverse transcriptase (Promega, Wisconsin, WI, USA). RT-qPCR was performed with GoTaq® qPCR Master Mix (Promega, USA) in a 20 μl reaction volume, and detection was performed using a DA7600 real-time nucleic acid amplification fluorescence detection system (Bio-Rad). The 2-ΔΔCt method was used to determine relative expression levels. We quantified GAPDH mRNA levels as an internal quantity control. All primers used for RT-qPCR were obtained from Sangon Biotech (Shanghai, China). RT-qPCR products were then subjected to electrophoresis. The primer sequences of target genes are listed in Table 2 .
6
Reverse Transcription and qPCR for Colon Cancer
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Colon cancer SW480 and T84 cells with different treatments were obtained and mRNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Subsequently, reverse transcription (42°C for 30 min followed by 85°C for 5 min) was performed using EasyScript Reverse Transcriptase (Beijing Transgen Biotech Co., Ltd.) followed by qPCR on a DA7600 Real-time Nucleic Acid Amplification Fluorescence Detection System (Bio-Rad Laboratories, Inc.) in a 25 µl volume system using the TransStart Green qPCR SuperMix (Beijing Transgen Biotech Co., Ltd.). The thermocycling conditions were as follows: Initial denaturation for 3 min at 95°C, followed by 35 cycles of denaturation at 95°C for 10 sec and annealing/extension at 55°C for 30 sec.
The primers were obtained from Sangon Biotech Co., Ltd. Melting-curve analysis was conducted to recognize the reaction specificity. All experiments were performed in triplicate and the data were analyzed using the 2−ΔΔCq method (30 (link)). Primers of RAGE, CDC4 and β-catenin were as follows: RAGE forward, 5′-GGG GTA CCA AGG AAG CAG GTA GGC AGC C-3′ and reverse, 5′-CCG CTC GAA TCC ATT CCT GTT CAT CTG C-3′; CDC4 forward, 5′-GTG GGA CAT ACA GGT GGA-3′ and reverse, 5′-CAA CGC ACA GTG GAA CTA-3′; β-actin forward, 5′-CAT GTA CGT TGCT ATC CAG GC-3′ and reverse, 5′-CTC CTT AAT GTC ACG CAC GAT-3′.
The primers were obtained from Sangon Biotech Co., Ltd. Melting-curve analysis was conducted to recognize the reaction specificity. All experiments were performed in triplicate and the data were analyzed using the 2−ΔΔCq method (30 (link)). Primers of RAGE, CDC4 and β-catenin were as follows: RAGE forward, 5′-GGG GTA CCA AGG AAG CAG GTA GGC AGC C-3′ and reverse, 5′-CCG CTC GAA TCC ATT CCT GTT CAT CTG C-3′; CDC4 forward, 5′-GTG GGA CAT ACA GGT GGA-3′ and reverse, 5′-CAA CGC ACA GTG GAA CTA-3′; β-actin forward, 5′-CAT GTA CGT TGCT ATC CAG GC-3′ and reverse, 5′-CTC CTT AAT GTC ACG CAC GAT-3′.
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