Genomic DNA of rice leaves was prepared by the method of CTAB (Allen et al., 2006 (link)). Total RNA of leaves was extracted by Easy Pure Plant RNA Kit (Trans Gene Biotech, Beijing, China). Quantitative RT-PCR procedures were carried out according to the manufacturer’s instructions by the Light Cycler System (Bio-Rad, Richmond, CA, United States) with the SYBR® qPCR Master Mix Kit (Trans Gene Biotech, Beijing, China) by a pair of primers (Supplementary Table S1 ). The relative expression levels were calculated by the 2-ΔΔCT formula (Livak and Schmittgen, 2001 (link)) using the reference gene (accession: XM_015774830) as an internal standard.
Light cycler system
Manufactured by Bio-Rad
Sourced in United States, China
The Light Cycler System is a real-time PCR instrument designed for rapid and sensitive DNA and RNA analysis. It utilizes a unique thermal cycling technology to enable fast and precise temperature control. The system is capable of performing quantitative and qualitative PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Easypure plant rna kit, by Transgene (2 mentions)
Sybr premix ex taqtm kit, by Takara Bio (2 mentions)
Superscript 3 rnase h reverse trancrip tase, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Easy spin kit, by iNtRON Biotechnology (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taqtm 2, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (1 mentions)
Primerscript rt reagent kit, by Takara Bio (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
9 protocols using light cycler system
1
Rice Genomic DNA and Gene Expression Analysis
2
Ginseng Transcriptome Analysis by qRT-PCR
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Total RNA was isolated from 200 mg of ground ginseng leaves using the Easy Spin kit (iNtRON Biotechnology, Korea) according to the manufacturer's instructions. For the first strand complementary DNA (cDNA) synthesis, reverse transcription was performed using iScriptTM cDNA synthesis kit (Bio-Rad, Korea) according to the manufacturer's instructions. qRT-PCR was performed with the LightCycler system (Bio-Rad) using SYBR Green Sensimix Plus Master Mix (Bio-Rad) under the following conditions: 15 min at 95°C; 40 cycles of 30 s at 95°C, annealing for 30 s for the different primers at 60°C, 30 s at 72°C, and then 7 min at 72°C. Data were subjected to an analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) statistics v. 18 (SPSS Inc., IBM, Armonk, NY, USA). The means were compared using Tukey's honest significant difference test at p < 0.05 to identify significant differences in levels of gene expression.
3
Quantitative RT-PCR Analysis of FoxP3 Gene
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To identify the expression level of FOX3 gene, the quantitative real time PCR method was used based on the light cycler system (bio-rad, USA). The reagent quantity and thermal condition of qRT- PCR were as similar as our previous study [27 (link)]. Briefly, 1 μg of isolated RNA was applied for cDNA synthesis kit (Fermentas, Italy) containing oligo dT primers and M-MLV reverse transcriptase in accordance with instruction’s guidline. GAPDH was tested as the reference gene. The primers for both described genes were listed in Table 1 . To amplify the FoxP3 gene, 0.5 μl of each cDNA was added to 20 μl master mixture solution including 2x cyber green solution and 0.5 μM of described primers. The PCR program was set up as an initial denaturation step of 4 min at 95 °C, 35 cycles of 20 s at 94 °C, 61 °C at 30 s as annealing tempreature for each gene and 20 s at 72 °C. The efficiency of all run was between 95% to 99%. The relative expression of FoxP3 gene mRNA was evaluated according to standard curve obtained from the specific target and housekeeping gene. Finally, Pfaffl method was applied for data analysis.
4
Quantifying Transcript Levels via qRT-PCR
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Total RNAs of leaves were extracted by Easy Pure Plant RNA Kit (TransGene Biotech, Beijing) and then determined using Nano-Drop2000 spectrophotometer. First-strand cDNA was prepared using SuperScript III RNase H–Reverse Trancrip-tase (Invitrogen, USA) using 2 μg of total RNA. Quantitative RT-PCR procedures were carried out by the Light Cycler System (Bio-Rad, Richmond, CA) with the SYBR® Premix Ex TaqTM Kit (TaKaRa, Dalian, China), in a 15 μL reaction solution containing 7.5 μL of SYBR® Premix Ex TaqTM 2×, 0.3 μmol·L–1 of each forward and reverse primers (Table S1 ), and 1.5 μL of cDNA template, and appropriate amounts of sterile ddH2O. All qRT-PCR amplification conditions were standardized according to the manufacturer’s instructions. Three independent replicates were performed on each cDNA sample. The relative expression level was calculated using the 2−△△Ct formula39 (link) with the reference genes β-actin gene (GenBank ID: AB181991.1) as internal standard.
5
Quantitative PCR Analysis of PyMYB10 and PybHLH
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qPCR DNA amplification was performed using the Light Cycler System (Bio-Rad, USA). All reactions were carried out in triplicate using a volume of 20μ Lreaction mixture containing 2μL of Master Mix (TaKaRa), 0.5 M of each primer, 2μl of diluted cDNA. The qPCR reaction programs were as follows: 95°C for 5 min; 40 cycles for 10 s at 95°C, 30 s at 56°C and 30 s at 72°C; and a final extension at 72°C for 3 min. The primers used for PyMYB10.1, PyMYB10 and PybHLH are listed in S1 Table . PyActin (accession number CN938023) was used as a constitutive control gene.
6
Liver Gene Expression Profiling
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Total-RNA was extracted from livers using an easy-BLUE total-RNA extraction kit according to the manufacturer’s instructions. Single-strand cDNA synthesis was performed using the Quantitact reverse transcription kit according to the manufacturer’s instructions. The RT-PCR analysis was performed with a QuantiTect™ SYBR Green PCR. The RT-PCR data were based on SYBR green amplification. The primer sequences are listed in Table 3 . mRNA was detected for PPARα, PPARγ, SREBP-1c, Fas, C/EBPα, and GAPDH using the LightCycler system (Bio-Rad, Hercules, California, U.S.A.). Each sample was run and analyzed in duplicate.
7
Quantification of HBV DNA and Gene Expression
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Total RNA was extracted from Hepg2.2.15 cells using Trizol (Invitrogen, USA) and the cDNAs were synthesized using PrimeScript™ RT Reagent Kit (TaKaRa, DaLian, China) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using a Light Cycler system (Bio-Rad Laboratories, Inc., Hercules, CA) using the SYBR Premix Ex Taq™ II Kit (TaKaRa, DaLian, China). The forward and reverse primers are shown in Table 1 . Quantification of HBV DNA in the supernatant was performed using quantitative real-time PCR kits (Da-an, Guangzhou, China) according to the instructions.
8
Analyzing Moco and Molybdenum Enzyme Genes
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To analyze the
expression of genes related to the synthesis of Moco and molybdenum
enzymes including CNX1, CNX2, CNX3, AO, XDH, and NR, fresh roots and
shoots were ground to a fine powder in liquid nitrogen. Total RNA
was extracted using TRIzol Reagent (Invitrogen, USA). The RNA concentration
was determined by a Nano-Drop2000 spectrophotometer (Thermo, USA)
and cDNA was synthesized using 20 μg of RNA and SuperScript
III RNase H–Reverse Trancrip-tase (Invitrogen, USA) according
to the manufacturer instructions. The SYBR Premix Ex TaqTM Kit (TaKaRa,
Dalian, China) and the Light Cycler System (Bio-Rad, Richmond, CA)
were used for RT-PCR. The actin gene was used as the internal standard.
Primer sequences for each gene are shown inTable S6 .
expression of genes related to the synthesis of Moco and molybdenum
enzymes including CNX1, CNX2, CNX3, AO, XDH, and NR, fresh roots and
shoots were ground to a fine powder in liquid nitrogen. Total RNA
was extracted using TRIzol Reagent (Invitrogen, USA). The RNA concentration
was determined by a Nano-Drop2000 spectrophotometer (Thermo, USA)
and cDNA was synthesized using 20 μg of RNA and SuperScript
III RNase H–Reverse Trancrip-tase (Invitrogen, USA) according
to the manufacturer instructions. The SYBR Premix Ex TaqTM Kit (TaKaRa,
Dalian, China) and the Light Cycler System (Bio-Rad, Richmond, CA)
were used for RT-PCR. The actin gene was used as the internal standard.
Primer sequences for each gene are shown in
9
Quantitative Gene Expression Analysis
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The samples were pulverized in liquid nitrogen and then thoroughly homogenized in 1 mL TRizol Reagent (Invitrogen, USA) per 100 mg sample. The total RNA was extracted according to the manufacturer's instructions. The concentration of extracted RNA was determined by a spectrophotometer (BioPhotometer, Eppendorf, Germany). First-strand cDNA was reversely transcribed using 2 μg of total RNA in a total volume of 20 μL extracts according to the manufacturer's protocol of PrimerScript RT reagent Kit (Takara, Japan).
Each PCR reaction was performed with 2 μL of the cDNA and 0.2 μmol/L of each primer in a Light-Cycler System with SsoAdvancedTM SYBR Green Supermix (Bio-Rad, USA). Briefly, the RT-PCR reactions were run at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 20 sec at 60°C in the CFX96 realtime system (Bio-Rad). Each sample was assayed in triplicate and the primer sequences, and PCR conditions were listed in Table 1. Preliminary experiments were performed with each pair of primers. Formation of the expected single product was ascertained in each reaction by melt curve analysis (starting from 72°C to 92.4°C). The gene expression level was determined using the delta Ct method (ΔCt), a variation of the Livak method, where ΔCt = Ct reference gene -Ct target gene .
Each PCR reaction was performed with 2 μL of the cDNA and 0.2 μmol/L of each primer in a Light-Cycler System with SsoAdvancedTM SYBR Green Supermix (Bio-Rad, USA). Briefly, the RT-PCR reactions were run at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 20 sec at 60°C in the CFX96 realtime system (Bio-Rad). Each sample was assayed in triplicate and the primer sequences, and PCR conditions were listed in Table 1. Preliminary experiments were performed with each pair of primers. Formation of the expected single product was ascertained in each reaction by melt curve analysis (starting from 72°C to 92.4°C). The gene expression level was determined using the delta Ct method (ΔCt), a variation of the Livak method, where ΔCt = Ct reference gene -Ct target gene .
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