A 2 μg of RNA sample from seedlings of N. benthamiana was incubated with DNase I (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions. The qRT-PCR protocol consisted of mixing 0.5 μL of DNase-treated RNA per reaction with Brilliant II SYBR Green QRT-PCR Master Mix Kit, 1-Step (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s instructions, and using a PCR protocol consisting in: an initial step of 50 °C for 30 min, a second step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 10 s. Fluorescence was measured at the last step. The relative expression of each gene was calculated according to the method described by Vandesompele et al. [38 (link)], using three reference genes (Table 2 ), and the seedlings without stimulus as normalizer. To detect a statistical difference, one-way ANOVA followed by Duncan’s multiple range test was realized at p < 0.01 using the software Statgraphics Centurion XV (Statgraphics.Net, Madrid, Spain).
Brilliant 2 sybr green qrt pcr master mix kit
Manufactured by Agilent Technologies
Sourced in United States
The Brilliant II SYBR Green QRT-PCR Master Mix Kit is a laboratory reagent designed for quantitative reverse transcription polymerase chain reaction (QRT-PCR) experiments. The kit includes a proprietary master mix formulation and necessary components to facilitate the detection and quantification of RNA targets.
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6 protocols using brilliant 2 sybr green qrt pcr master mix kit
1
Quantitative Gene Expression Analysis in Nicotiana benthamiana
2
Quantitative RT-PCR Analysis of Fructose Genes
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Determination of relative expression levels was performed on total RNA using the Stratagene MX3005P system, the Brilliant II SYBR green qRT-PCR master mix kit (Agilent), and primers specific to fruB, fruK, fruA, and 4.5S (Table S2 in the supplemental material). The reactions were set up in 96-well optical reaction plates and contained 1× Brilliant SYBR green qPCR master mix, 30 nM ROX reference dye, each primer at 100 nM, 100 ng RNA, and 1 μl RT/RNase block enzyme mixture in a 25-μl reaction mixture. The following conditions were used for cDNA synthesis and PCR: 30 min at 50°C, 10 min at 95°C, and 40 cycles of 30 s at 95°C and 1 min at 60°C (Agilent). MxPro QPCR software (v. 4.10) was used to determine threshold cycle (CT) values for each reaction, and relative RNA concentrations were calculated from the CT values by comparison to standard curves. All transcript levels were normalized to a 4.5S RNA endogenous control. No signals were detected in no-template controls and no-reverse transcriptase (RT) controls.
3
Quantitative RT-PCR Using Brilliant II SYBR
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qRT-PCR was carried out using a Stratagene MX3005P System and Brilliant II SYBR Green QRT-PCR Master Mix Kit (Agilent, 600835). The reactions contained 1× Brilliant SYBR Green QPCR Master Mix, 30 nM ROX reference dye, each primer at 100 nM (Table S2 ), 100 ng RNA, and 1 μL RT/RNase block enzyme mixture in a 25 μL reaction. All the reactions were carried out at the following conditions: 30 min at 50°C, 10 min at 95°C, and 40 cycles of 30 s at 95°C and 1 min at 60°C in 96-well optical reaction plates (Agilent, 401334). A dissociation curve analysis was carried out at the end of amplification to confirm PCR product specificity. Fluorescence data were collected at the end of the extension step. Technical replicates as well as no template and no RT negative controls were included and at least 3 biological replicates were studied in each case. No signals were detected in no-template controls and no-RT controls. Expression of RNA of interest was normalized to an endogenous control (4.5S RNA). Statistical analysis was performed using GraphPad Prism 5 software.
4
Quantitative Reverse Transcription-PCR Analysis
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RNA samples were used for quantitative reverse transcription-PCR (qRT-PCR) to quantify relative expression levels using a Stratagene MX3005P system, a Brilliant II SYBR green qRT-PCR master mix kit (Agilent), and primers specific to mtlA, mtlS, and 4.5S RNA. The reaction mixtures were set up in 96-well optical reaction plates and contained 1× Brilliant SYBR green qPCR master mix, 30 nM carboxy-X-rhodamine reference dye, each primer at 100 nM, 100 ng RNA, and 1 μl reverse transcriptase-RNase block enzyme mixture in a 25-μl reaction mixture. The following conditions were used for cDNA synthesis and PCR: 30 min at 50°C, 10 min at 95°C, and 40 cycles of 30 s at 95°C and 1 min at 60°C (Agilent). MxPro QPCR software (version 4.10) was used to determine the threshold cycle (CT) values for each reaction, and relative RNA concentrations were calculated from the CT values by comparison to standard curves. All transcript levels were normalized to a 4.5S RNA endogenous control. No signals were detected in the no-template controls and no-reverse transcriptase controls. Statistical analysis was performed using GraphPad Prism (version 7) software.
5
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated from cells using GeneMATRIX Universal RNA Purification Kit (EURx). Isolated RNA was quantified using BioPhotometer (Eppendorf). qRTPCR was performed using Brilliant II SYBR Green QRT-PCR Master Mix Kit (Agilent Technologies). qRT PCR normalization was conducted using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. qRTPCR reactions were performed on an Mx-300 PCR cycler (Stratagene) for 40 cycles. The thermal profile for the PCR reaction was as follows: 50°C for 30 min (reverse transcription); 95°C for 15 min (initial denaturation); 94°C for 15 s (denaturation), 60°C for 1 min (annealing), 72°C for 30 s (elongation); 72°C for 10 min (final elongation). The primer sequences used in the experiment are presented in Table I . The specificity of qRT PCR reaction was confirmed by melting curve analysis and by the use of electrophoresis on 1.2% agarose gels, stained with Gel Red fluorescent dye (Biotium). The relative gene expression was calculated using comparative 2(–ΔΔCt) [26 (link)] and with Mx3000p software (version 2.0).
6
SYBR Green QRT-PCR Assay Protocol
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Brilliant ®II SYBR Green QRT-PCR Master Mix kit (Agilent technologies) was used with 10 ng total RNA and 100 nM each of forward and reverse primer as listed in Additional file 1 : Table S3 (17–22). New primers were designed using Primer3.
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