Total RNA was isolated and purified from treated leaves according to the protocol of RNeasy® plant mini kits (Qiagen). The expressions of selected genes were analyzed by real-time quantitative reverse transcriptase (RT-PCR) using the fluorescent intercalating dye SYBRGreen with a detection system (Opticon 2, MJ Research, Waltham, MA, United States). The VTC2 coding mRNA sequence was amplified with the following primers: forward (5′-ACCTTCACAGGAGGATGCTG-3′) and reverse (5′-CCACGCGGAACTCTGTAGGG-3′). The VTC4 coding mRNA sequence was amplified with the following primers: forward (5′-GACCACAAGTTCATCGGCGAG-3′) and reverse (5′-CCACGAACAGCTGTGAAAAGCT-3′) (Hu et al., 2014 (link)). eEF1A (s) was used as a standard control for RT-PCR. The two-step RT-PCR procedure was performed following the methods we described earlier.
Opticon 2
Manufactured by Bio-Rad
Sourced in United States
The Opticon 2 is a real-time PCR detection system designed for researchers. It is capable of monitoring fluorescence during the amplification of DNA samples, enabling the quantification of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (11 mentions)
Rneasy isolation kit, by Qiagen (6 mentions)
Rneasy plant mini kit, by Qiagen (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Gotaq one step rt qpcr kit, by Promega (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Bio-Rad (1 mentions)
Omniscript rtase, by Qiagen (1 mentions)
Trizol extraction method, by Thermo Fisher Scientific (1 mentions)
Rnaeasy, by Qiagen (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
45 protocols using opticon 2
1
Quantifying Plant Gene Expression
2
Transcriptome Analysis of Brassica napus Silique Development
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The siliques were collected at different stages, which is based on the results of emasculation, bagging and artificial pollination when the flower was ready to bloom. The siliques were gently grinded in liquid nitrogen, and then the seeds were picked out in liquid nitrogen using tweezers for RNA extraction. Total RNA was extracted from 10, 15, 20, 25, 30, 35, 40, 45 and 50 DAP (days after pollination) seeds and silique walls, and purified using RNeasy Mini kit (Qiagen, German).
The expression of genes was analyzed by real-time quantitative RT-PCR using the fluorescent intercalating dye SYBR Green in a detection system (MJ Research, Opticon 2). The BnActin2 (BnACT2) gene was used as standard control in the quantitative RT-PCR reactions. Two-step RT-PCR procedure was performed using a method described previously [44 (link)]. The genes detected by RT-PCR were analyzed by basic local alignment search tool (http://www.genoscope.cns.fr/blat-server/cgi-bin/colza/webBlat ), and multiple sequences alignment of homologous genes was completed by Clustal W. Then, the gene-specific primers were designed based on multiple sequences alignment (S1 Table ).
The expression of genes was analyzed by real-time quantitative RT-PCR using the fluorescent intercalating dye SYBR Green in a detection system (MJ Research, Opticon 2). The BnActin2 (BnACT2) gene was used as standard control in the quantitative RT-PCR reactions. Two-step RT-PCR procedure was performed using a method described previously [44 (link)]. The genes detected by RT-PCR were analyzed by basic local alignment search tool (
3
Real-Time Quantitative PCR Protocol
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The real-time quantitative PCR (qRT-PCR) was performed using the fluorescent intercalating dye SYBR Green in a detection system (MJ Research, Opticon 2), and MiACT was used as a standard control (Luo et al., 2013 (link)). The two-step RT-PCR procedure was performed according to the method described by Li et al. (2005) (link).
4
Quantifying Wheat and Tobacco Stress Responses
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Expressions of TaPP2C1 in wheat and stress/ABA-responsive genes in transgenic tobacco plants were determined with qRT-PCR method in MJ Research Opticon 2 instrument. Primers (P3-P14 in S1 Table ) used in qRT-PCR detection had high specificity and efficiency according to agarose gel electrophoresis, melting curve and sequence analyses. Prior to actual experiments, several primer and template dilutions were performed to test the appropriate primer and template concentration. In all of the experiments, each sample contains four replicates and possible contamination was excluded by appropriate negative controls. NtUbiquitin and TaActin were used as reference gene for tobacco and wheat respectively to normalize the transcripts of target genes. The relative expression levels of the target genes were assessed according to the 2-ΔΔCt method [16 (link)].
5
RNA Isolation and Real-Time PCR Analysis
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RNA was isolated from cells using the TRIzol extraction method (Invitrogen) and 40 μg of purified RNA used for reverse transcription using Omniscript RTase (Qiagen) for 1 h at 37 °C. cDNAs were then subjected to real-time PCR using DyNAmo Flash SYBR Green (Finnzymes). Amplification was performed using an Opticon 2 thermocycler (MJ Research) and data was analyzed using the comparative Ct method.
6
p53 gene expression profiling
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P53 RT2 Array (QIAGEN, Germantown, MD) were purchased and used according to the manufacturer’s guidelines. ALL cell lines were subjected to 100 nM YM155 for 24 h, and RNA was extracted from the cell line using RNAeasy (QIAGEN). One microgram of RNA was used to make first strand cDNA using RT2 First Strand kit. Quantitative PCR was performed on Opticon 2 (MJ Research). Generated data was then uploaded to the RT2 Array website.
7
Quantifying NMDA and Cytochrome Gene Expression
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Frontal cortex, temporal cortex and hippocampus were grinded under powder based on a cold-milling process to homogenized tissues. Purification of mRNA was then performed on 10 mg of powder by the means of a μMACSTM mRNA isolation kit (Miltenyi Biotec, Leiden, The Netherlands). Superscript II (Thermo Fisher Scientific, Merelbeke, Belgium) was used for first-strand cDNA synthesis as previously described [30 (link)]. Quantitative PCR amplification was carried out in an Opticon 2 (MJ Research, Bio-Rad, Nazareth, Belgium) using Platinum Taq DNA polymerase (Thermo Fisher Scientific, Merelbeke, Belgium). Primers and PCR conditions were as previously described for NMDA [6 (link)] and Cyp1a1/1b1 [30 (link)]. We have previously confirmed the stability of Gapdh as reference gene in brain [31 (link)]. RT-qPCR results for Nmda subunits (-R1, -R2A and -R2B) were as gene copies/102Gapdh copies. Regardless of accurate fragment size, the limit of detection was established at 38 cycles. For the cytochromes Cyp1a1 and Cyp1b1, the gene expression was normalized to those of Gapdh/Ppia and expressed using the 2-ΔΔCt method [32 (link)].
8
Quantifying Gene Expression in Arabidopsis
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Total RNA was extracted from Arabidopsis seedlings with Trizol reagent (Invitrogen) with Trizol reagent (Invitrogen) according to the manufacture protocol, and treated with DNase I (Takara) at 37 °C for 1 hour to eliminate genomic DNA contamination. The cDNA was reversely synthesized from the treated RNA, and used as real-time PCR templates with gene-specific primers. The PCR amplification was performed in the detection system (Opticon2; MJ Research, New Haven, Connecticut, USA) according to the method described earlier58 (link), using AtACTIN2 gene as an internal reference. Each qRT-PCR analysis was repeated three times. Mean values and standard variations were estimated from the data of three biological experiments. All primers used in the experiments are listed in Table S1 .
9
Quantitative Real-Time PCR for Gene Expression Analysis
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The total RNA was isolated from 100mg of the leaf tissue collected from the respective stress treatments according to a modified protocol [13 ]. About 3μg of total RNA was reverse transcribed to synthesize first strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The cDNA was used as the template for expression analysis and the house keeping gene, elongation factor (elf) was used as the internal control. The qRT-PCR was performed in a q-PCR machine (Opticon2, MJ Research, USA), with the fluorescent dye SYBR-green (SYBR Premix Ex Taq, Perfect Real Time, Takara, Japan) under standardized PCR conditions using target specific primers as listed (S1 File ). The relative transcript level was calculated from three independent replications; calculated using comparative ΔCt method [24 (link)] and student t-test was performed (p = 0.05).
10
Validating RNA-Seq Findings by qPCR
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To validate the findings of the RNA-Seq assay, 20 DEGs were randomly chosen and their relative expression confirmed by qPCR analysis using the fluorescent intercalating dye SYBRGreen in the Opticon 2 detection system (MJ Research, Waltham, MA, USA). Details of the selected genes and the respective primers are listed in Table S1 . Gene expression levels were normalized against the hulless barley gene HvADP (Ferdous et al., 2015 (link)) and calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). Three technical replicates were generated for each biological sample.
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