Sixty-day-old plants grown in the early season and the late season were used for the expression analysis of the flowering-time genes. Leaves were harvested every 4 h within 1 day (7-time-points). Total RNAs were extracted using an Eastep®Super Total RNA Extraction Kit (Promega, Shanghai, China) and reverse-transcribed into cDNA using a Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China) kit. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays were performed using a PerfecStart Green qPCR SuperMix (Transgen, Beijing, China) kit on a C1000 CFX96 Real-time PCR Detection System (Bio-rad, Hercules, CA, USA) with the following setting: 95 °C for 2 min, 42 cycles of two-step amplification (95 °C for 30 s, 60 °C for 30 s). PCR was repeated three times for technical repetition. The primers used for gene expression analysis are listed in Table S3 .
C1000 cfx96 real time pcr detection system
Manufactured by Bio-Rad
Sourced in China
The C1000 - CFX96 Real-Time PCR detection system is a lab equipment product designed for real-time PCR analysis. It features a thermal cycler and a fluorescence detection system for quantitative gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Eastep super total rna extraction kit, by Promega (1 mentions)
Transcript one step gdna removal and cdna synthesis supermix, by Transgene (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Rt pcr grade water, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Sensifast sybr no rox one step mix, by Meridian Bioscience (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
All in one first strand cdna synthesis kit, by GeneCopoeia (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
8 protocols using c1000 cfx96 real time pcr detection system
1
Diurnal Gene Expression Profiling in Flowering Plants
2
Thermal Stability Analysis of Ric-8A Proteins
3
Thermal Stability Analysis of Ric-8A Proteins
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Thermal stability of indicated Ric-8A proteins were analyzed by a SYPRO Orange fluorescent dye-based denaturation assay using a Bio-Rad C1000 – CFX96 Real-Time PCR detection system. SYPRO Orange dye was added at 1000x dilution in samples containing 0.5mg/mL Ric-8A 1–452 or full-length with or without phosphorylation (Buffer: 20 mM HEPES pH:7.4, 140 mM KCl, 5% glycerol and 1mM TCEP). Fluorescence of SYPRO orange is measured between 10 to 80 C (30 s per 1 C increments) using the SYBR channel on the CFX96 plate reader.
4
Quantitative PCR Analysis of siRNA Knockdown
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After two days of treating the cells with SiNCs containing siRNA as described earlier, the cells were collected and the total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). The RNA concentration of the extracted RNA samples was measured using a Nanodrop (NanoDrop™ 8000 Spectrophotometer, Thermo Fisher Scientific, Wilmington, NC, USA). RT–PCR grade water (Invitrogen) was used throughout the experiment. Then, the cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-Rad) and the qPCR reaction was performed using the iQ™ SYBR® Green Supermix (Bio-Rad) in a Thermal cycler (C1000, CFX96 Real-Time PCR detection system, Bio-Rad). The qPCR data was analyzed using the 2−ΔΔCT method. The results were expressed as a relative expression of two independent experiments, compared to the results of the control cell.
5
cDNA Synthesis and qPCR Analysis
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cDNA was synthesized from total RNA using the SuperScript III first-strand synthesis system (Life Technologies, Inc.) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using the Power SYBR green PCR master mix (2×; Life Technologies, Inc.) on a CFX96-C1000 real-time PCR detection system (Bio-Rad) according to the manufacturers’ instructions. Primer sequences can be found in Text S1 in the supplemental material.
6
Optimized Real-time RT-PCR Protocol
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Quantitative real-time RT-PCR conditions were optimised with GI.1c in vitro transcripts. Reactions were performed in a final volume of 10μl and contained 1× SensiFAST SYBR No-ROX One-Step mix (Bioline, Alexandria, NSW), 0.5 μM of each primer, 0.2 μl of RNase inhibitor, 0.1 μl of reverse transcriptase, and 1 μl of template RNA. Cycling was performed using a CFX96 C1000 real-time PCR detection system (Bio-Rad Laboratories, Gladesville, NSW), with reverse transcription conducted at 45 ºC for 10 min, followed by denaturation at 95 ºC for 5 min, and then 40 cycles of 95 ºC for 10 s, 63 ºC for 40 s, 78 ºC for 10 s with data acquisition. Melt curve analysis was conducted at 65-95 ºC in 0.5 °C increments at 5 s per increment. Annealing temperature was optimised by gradient PCR.
Data were analysed using CFX manager 3.1 software (Bio-Rad Laboratories, Gladesville, NSW) using a baseline threshold of 200 and baseline subtracted curve fit setting. Average assay efficiency was 95%. Amplicons were separated on 2% agarose gels and visualised by staining with SYBRsafe DNA gel stain (Life Technologies, Scoresby, VIC) to verify that products were of the expected size.
Data were analysed using CFX manager 3.1 software (Bio-Rad Laboratories, Gladesville, NSW) using a baseline threshold of 200 and baseline subtracted curve fit setting. Average assay efficiency was 95%. Amplicons were separated on 2% agarose gels and visualised by staining with SYBRsafe DNA gel stain (Life Technologies, Scoresby, VIC) to verify that products were of the expected size.
7
RT-qPCR Analysis of HMGB1 and RAGE Expression
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Total tissue RNAs were extracted from the separated tissues, which were then homogenized on ice using TRIzol® reagent (Thermo Fisher Scientific, Inc.). The RNA concentration was detected with a GeneQuant 1300 spectrophotometer (Cytiva) according to the manufacturer's protocols. A 1-µl sample of total RNA was reverse-transcribed into cDNA in a 20 µl system using an All-in-One First-Strand cDNA Synthesis kit (GeneCopoeia, Inc.) based on the manufacturer's protocols. Then RT-qPCR was performed using a SYBR-Green qPCR kit (cat. no. F-416L; Finnzymes; Thermo Fisher Scientific, Inc.) on a CFX96 Real-time PCR Detection System C1000 (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: 95°C for 2 min; followed by 40 cycles of 95°C for 10 sec, 60°C for 34 sec and 72°C for 33 sec. The primers used for RT-qPCR were synthesized by Takara Biotechnology Co., Ltd. and the gene expression was quantitated by the 2−ΔΔCq method (26 (link)) from 3 repeated experiments. The primer sequences were as following: GAPDH forward, 5′-CCTCGTCTCATAGACAAGATGGT-3′ and reverse, 5′-GGGTAGAGTCATACTGGAACATG-3′; HMGB1 forward, 5′-TGTTCTGAGTACCGCCCAAA-3′ and reverse, 5′-CTTGGCGGCCTTCTTTTCAT-3′; and RAGE forward, 5′-TCACAGAAACCGGTGATGAAG-3′ and reverse, 5′-CTCGAGTCTGGGTTGTCGTT-3′.
8
Quantifying Lipid and Aquaporin Metabolism
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Real-time quantitative PCR was performed using PowerUp™ SYBR ® Green Master Mix (Life Technologies) and Xpert Fast SYBR (GRiSP) to amplify AQP (AQP3, 5 and 7) and markers of lipid metabolism (ADIPO, LEP, GLUT4, PPARα and PPARγ), respectively. The final reaction volume of 20 μl was prepared using 10 μl of SYBR master mix, 3 μl of template cDNA, 2 μl of forward and reverse primers and 3 μl of molecular-biologygrade water. The reaction was performed on a CFX96™ Real-Time PCR Detection System C1000 (BioRad) consisting of an initial denaturation step at 95°C for 3 min, forty-five cycles of denaturation at 95°C for 10 s and annealing/extension at 59 or 62°C (for AQP and markers of lipid metabolism, respectively) for 30 s. The relative expression levels were normalised to reference genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) for AQP3, AQP5 and AQP7; ribosomal protein L27 (RPL27) for ADIPO, LEP, GLUT4, PPARα and PPARγ) and calculated using a variation of the Livak method (37) , corrected for variation in amplification efficiency, as described by Fleige & Pfaffl (38) .
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