Total RNAs of leaf, root, stem, anther, pistil, petal, sepal, pollen, and pollen tube were extracted using TRI Reagent Solution (Ambion), and total RNAs of seeds at different stages were extracted with RNAqueous™ (Ambion). All total RNAs were treated with RNase-free DNase I (Promega) and cDNAs were synthesized using ReverTra Ace (Toyobo) under the conditions recommended by the manufacturer. mRNA isolation from sperm cells, egg cells, zygotes, apical cells, basal cells, and embryos at different stages and cDNA synthesis were performed according to a previous procedure (Ma et al., 2011 (link); Xin et al., 2011 (link); Zhao et al., 2011 (link)). Quantitative real-time reverse transcription–PCR (RT–qPCR) was conducted for cystatin gene expression pattern analysis. RT–qPCR was performed in a 20 μl reaction mixture containing 10 μl of 2×FastStart Universal SYBR Green Master (Roche), 250nM of each primer (Supplementary Table S1 at JXB online), and cDNA prepared from different tissues. Conditions for RT–qPCR were as follows: activation of FastStart Taq DNA polymerase at 95 °C for 10min, and >40 cycles (95 °C for 15 s and 60 °C for 1min) with a Rotor-Gene 6000 system (Corbett Research). The data analysis was conducted according to a previous procedure (Ma et al., 2011 (link)).
Faststart universal sybr green master
Manufactured by Roche
Sourced in United States, Switzerland, Germany, China, Japan, Canada, Spain, United Kingdom, Australia
FastStart Universal SYBR Green Master is a ready-to-use PCR reaction mix that contains all the necessary components, including SYBR Green I dye, for real-time quantitative PCR (qPCR) analysis. It is designed to provide reliable and consistent results for a wide range of sample types and target sequences.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (334 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (112 mentions)
Trizol, by Thermo Fisher Scientific (107 mentions)
Rneasy mini kit, by Qiagen (76 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (70 mentions)
Primescript rt reagent kit, by Takara Bio (61 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (54 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (41 mentions)
Primescript rt master mix, by Takara Bio (30 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (29 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (28 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (22 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (20 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (20 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (20 mentions)
Trizol reagent, by Takara Bio (19 mentions)
Rneasy plus mini kit, by Qiagen (19 mentions)
Dnase 1, by Thermo Fisher Scientific (19 mentions)
M mlv reverse transcriptase, by Promega (17 mentions)
Iscript cdna synthesis kit, by Bio-Rad (17 mentions)
1 070 protocols using faststart universal sybr green master
1
Comprehensive Plant Transcriptome Analysis
2
qRT-PCR Validation of RNA-seq Analysis
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qRT-PCR was used to validate the gene expression levels determined by RNA-seq analysis. Nineteen RNA samples were randomly selected from the samples subjected to RNA-seq analysis, and cDNA libraries were constructed using the first-strand cDNA synthesis with the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). Seventeen genes were selected, and primers were designed using the Primer Express 3.0 software (Applied Biosystems) (Supplemental Table S4 ). The qRT-PCR was performed using a Roche LightCycler480 II real-time PCR system (Roche). Each reaction contained 10 μL of 2 × FastStart universal SYBR Green master (Roche), 2.0 μL diluted cDNA, and 0.8 μL each of the forward and reverse primers in a final volume of 20 μL. The PCR conditions consisted of predenaturation for 5 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 25 sec at 60°C, and 25 sec at 72°C. At the end of the PCR cycles, a melting curve analysis was performed to validate the specificity of the PCR product. The housekeeping gene LYS14 was used as a reference for normalization, and three replicates were performed for each cDNA sample. Data analysis was performed as reported previously (Livak and Schmittgen 2001 (link)).
3
Quantitative RT-PCR Analysis of C2C12 Cells
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Quantitative RT-PCR was performed as previously described 11 (link). Briefly, differentiated C2C12 cells were treated with 10 µg/mL TG for 24 h. The total RNA was extracted with TRIzol (Invitrogen) reagent and reverse-transcribed into cDNA. RT-qPCR was performed using a 2×FastStart Universal SYBR GreenMaster (Roche Diagnostics, Mannheim, Germany) system according to the manufacturer's protocol. The PCR primer sequences were listed in Table 1 . Relative quantification of the expression of each gene was calculated using the ΔΔCT method.
4
Quantitative Gene Expression Analysis
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Quantitative reverse transcription-PCR (qRT-PCR) was performed on the ABI PRISM 7500 Real Time System (Applied Biosystems, Foster City, CA, USA) to determine the expression patterns of PHB, CAPZB, and TEKT2 genes by using specific primers as provided in Table 1 . The reaction system was 20 μL with 1 μL cDNA, 10 μL 2×FastStart Universal SYBR Green Master, 0.4 μL ROX Reference Dye (Roche, Basel, Switzerland), 0.6 μL primer forward/reverse (10 nmol/L), 8.0 μL RNase-Free H20. Reaction conditions were as follows: 95°C for 10 min; 95°C for 15 s, 60°C for 1 minute for 40 cycles. Each sample was repeated 3 times. ACTB was used as the reference gene. The 2−ΔΔCT method was used to calculate the relative expression of each gene.
5
Validating RNA-Seq Gene Expression by qRT-PCR
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Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the gene expression levels of DEGs detected by RNA-Seq. Total RNA from the same samples as used for cDNA library construction was used for first-strand cDNA synthesis with the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, China). Primers were designed using Primer Express 3.0 software (Applied Biosystems, USA) and are listed in Additional file 7 Table S1. The qRT-PCR was performed using a Roche LightCycler480 II real-time PCR system (Roche, Germany). Each reaction contained 10 μL of 2 × FastStart Universal SYBR Green Master (Roche, Germany), 2.0 μL diluted cDNA, and 0.8 μL each of the forward and reverse primers in a final volume of 20 μL. The PCR conditions consisted of pre-denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 25 s, and 72 °C for 25 s. At the end of the PCR cycles, melting curve analysis was performed to validate the specificity of the PCR product. The maize GAPDH gene (accession number: NM_001111943.1) was used as an internal control for normalization, and three technical replicates of each cDNA sample were performed for qRT-PCR analysis. Data analysis was performed as reported previously [42 (link)].
6
Quantitative Analysis of mRNA Expression
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The methods, including PCR protocol and data calculation, were described previously (Chang et al., 2016; Hu et al., 2017) . Expression levels of target genes were quantified using 2× FastStart Universal SYBR Green Master (04913850001; Roche, Mannheim, Germany) with the MiniOpticon real-time PCR system (Bio-Rad). Then, mRNA values for the target genes were normalized using the expression of β-actin mRNA from the same cDNA sample (Tang et al., 2010) . Primer information used is listed in Table S2. The amplification efficiency was similar for all primer pairs used in this study (range: 0.93-1.08). To confirm the specificity of these amplifications from each test sample, sequencing, melting curve analysis, and electrophoresis were carried out. For each unknown sample, relative fxyd gene expression was obtained using the formula 2^-[(Ct target genes,n -Ct β-actin,n ) -(Ct target genes,c -Ct β-actin,c )], where Ct corresponds to the threshold cycle number (Livak and Schmittgen, 2001) . The letter "n" indicates each cDNA sample used in these experiments and "c" indicates the control mixed with cDNAs from many organs.
7
RNA Extraction and Quantitative PCR Analysis
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Total RNAs were extracted with TRIzol reagent (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. Afterward, 1 μg of DNase-treated RNA was reverse-transcribed using a PrimeScriptTM 1st Strand cDNA synthesis kit (Takara) according to the manufacturer’s protocol. The qPCR analysis was conducted with FastStart Universal SYBR Green Master (Roche), and reactions were performed in an ABI 7900HT (Applied Biosystems) at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 58°C for 30 s. The internal controls were Tubulin and Actin2 for Arabidopsis and rice, respectively. Each sample was analyzed in triplicate and relative amounts of mRNA were calculated per the comparative threshold cycle method.
For semi-quantitative RT-PCR, two-step RT-PCR method was chosen. Total RNA extractions and cDNA synthesis were performed as described above. All PCR amplifications were conducted in a total volume of 20 μL under the following conditions: 22–27 cycles of denaturation (94°C, 30 s), annealing (58°C, 35 s), and extension (72°C, 30 s). The primer pairs for semi-quantitative RT-PCR and qPCR are listed in Supplementary TableS1 .
For semi-quantitative RT-PCR, two-step RT-PCR method was chosen. Total RNA extractions and cDNA synthesis were performed as described above. All PCR amplifications were conducted in a total volume of 20 μL under the following conditions: 22–27 cycles of denaturation (94°C, 30 s), annealing (58°C, 35 s), and extension (72°C, 30 s). The primer pairs for semi-quantitative RT-PCR and qPCR are listed in Supplementary Table
8
Quantitative Analysis of miRNA Expression
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Total RNAs from hMSCs were isolated by TRIZOL Reagent (Invitrogen, USA) following the manufacturer’s instructions. After RNA extraction, RNA samples were reversely transcribed by High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). As for the detection of miRNA, the NCode miRNA First-Strand cDNA Synthesis kit (Invitrogen, USA) was applied in our study. Then, the Fast start Universal SYBR Green Master (Roche, USA) was used for the quantitative Real Time–PCR (qRT-PCR). The primer sequences are available in Supplementary Table 1. The relative expression levels of target genes were normalized to housekeeping gene RPLPO and analyzed by 2−ΔΔCT method.
9
ChIP-qPCR Analysis of H3K9me3
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ChIP was performed with the iDeal ChIP-qPCR kit (Diagenode, #C01010180) according to the recommendation of the manufacturer. Briefly, mESC cells were crosslinked in 1% formaldehyde (Euromedex, #EM-15686) for 15 min at room temperature and quenched with 0,12 M glycine provided in the kit. The extracted chromatin was sonicated with a Bioruptor Pico (Diagenode) and immunoprecipitation was performed using 1ug of an anti-H3K9me3 (Abcam, #ab8898) and 1ug of a rabbit IgG-isotype (Diagenod, #C15410206) control antibody. Eluted DNA was quantified by real-time PCR on a Roche LightCycler by using FastStart Universal SYBR Green Master (Roche, #4913850001) with the set of primers listed in Supplementary Data 7 . Data were normalized with respect to percentage of input and correspond to the mean +/− SD from at least three replicates.
10
Quantifying DNMT Expression in PBMCs
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