RNA was extracted from the leaves of 1-month old plants grown in long-day conditions in soil or in hydroponics (as specified in the figure legend), using the RNeasy RNA Plant kit (Qiagen). A total of 0.5-1 μg RNA was reverse-transcribed into cDNA with Superscript II reverse transcriptase (Thermo Fisher Scientific) and d(T)20 oligomers (IDT). To determine GGLT1 and GFT1 expression, the resultant cDNA was subsequently used as the template in a qPCR reaction containing 2× QuantiFast SYBR Green PCR Kit Master Mix (Qiagen) (plants grown in soil) or 2× SYBR Select Master Mix (Applied Biosystems) (plants grown hydroponically) and gene specific primers for GGLT1 and GFT1. Expression of Arabidopsis PP2A (At1g13320) and a SAND family gene (At2g28390) was quantified (see Supplemental Table S2 for all primer sequences), as they have previously been established as housekeeping genes (Czechowski et al., 2005 (link)). The PCR reactions were performed using the CFX96 Touch System real-time PCR detection system (Biorad) and the following thermal profile was used for all PCR reactions: 95 °C for 5 min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s.
Cfx96 touch system real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The CFX96 Touch System is a real-time PCR detection system manufactured by Bio-Rad. It is designed for quantitative PCR (qPCR) analysis. The system includes a thermal cycler, a detection unit, and a touchscreen interface for instrument control and data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy plant rna kit, by Qiagen (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Quantifast sybr green pcr kit master mix, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qpcr plate, by Bio-Rad (1 mentions)
Sybr green, by Agilent Technologies (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
3 protocols using cfx96 touch system real time pcr detection system
1
Quantifying Gene Expression in Arabidopsis
2
Quantitative RT-PCR Analysis of Ral GTPases
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RNA from UB RalA KD cell line, UB RalB KD cell line and UB RalCtl KD cell line was extracted using RNeasy kit (Cat # 74106, Qiagen, Germany) by following the manufacturer´s guideline. Complementary DNA (cDNA) was prepared using 1 μg RNA according to the guideline of first strand cDNA Synthesis Kit (Cat # K1612, ThermoScientific). In quantitative polymerase chain reaction (qPCR), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control (housekeeping) gene. Master-mix for primers was prepared using 5 μM of combined forward and reverse primer, 2.2 μL of nuclease free water and 5 μL of SYBR Green (Cat # 600883, Agilent Technologies, United States). Next, 8 μL of master-mix and 2 μL of cDNA were mixed in a well in 96-well plate of qPCR (Bio-Rad, United Kingdom). qPCR Plate was processed to CFX96 Touch System Real-Time PCR Detection System (Bio-Rad, United states). Gene expressions were detected using the program: 95 °C for 10 min, 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s x (40 cycles), followed by 55 °C for 5 s and 95 °C for 5 s (melting curve). qPCR data was analyzed using delta delta Ct (∆∆Ct) formula to determine the gene expression (mRNA level). Primers for GAPDH, RalA and RalB were designed using Primer3web version 4.1.0. Primers used for different genes in qPCR experiment are listed in Supplementary table 3 .
3
Quantitative Gene Expression Analysis
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Total RNAs were extracted from cell cultures and mice tissues using TRIzol (Thermo Fisher Scientific, Inc.). cDNAs were synthesized using M-MuLV Reverse Transcriptase in the QuantiTect® Reverse Transcription kit (Qiagen, Inc.) for 15 min at 42°C. qPCR was performed on a CFX96 Touch™ System Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), using SYBR-Green (Takara, Bio, Inc., Otsu, Japan). The thermocycling conditions were as follows: 40 Cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 15 sec and extension at 72°C for 10 sec. The relative amount of transcripts was calculated using the 2−ΔΔCq method (28 (link)). GAPDH and RNU6-1 were used as housekeeping genes. SOCS1 was normalized to GAPDH and miR-155 was normalized to RNU6-1. All primers used for the RT-qPCR analysis are listed in Table II .
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