Approximately 4 μg of total RNA was reverse-transcribed to cDNA using the HiScript® II Reverse Transcriptase system (Vazyme Biotech Co., Ltd., Nanjing, China). The quantitative real-time PCR (qRT-PCR) was performed in 20 μL reaction mixture with the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 10 s. The Actin gene was used as an internal control. The primers were designed using Primer Premier 5 and listed in Table S6 . The levels of gene expression were calculated by using the comparative 2−ΔΔCT value method [38 (link)].
Hiscript 2 reverse transcriptase system
Manufactured by Vazyme
Sourced in China
The HiScript® II Reverse Transcriptase system is a tool for converting RNA into complementary DNA (cDNA) through the process of reverse transcription. It is a high-performance reverse transcriptase enzyme that enables efficient and reliable cDNA synthesis from various RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 real time system, by Roche (6 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (3 mentions)
Transzol plant kit, by Transgene (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Sybr green mix, by Roche (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
9 protocols using hiscript 2 reverse transcriptase system
1
qRT-PCR Gene Expression Analysis Protocol
2
Transcriptional Analysis of Rice Development
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Total RNA was extracted from various plant tissues of Nipponbare, as well as from developing grains (0, 7, 14, 21, 28, 35 and 42 days after flowering; DAF) and germinating seeds (4‐, 8‐, 12‐, 18‐, 24‐, 36‐ and 48‐h imbibition), using the TransZol Plant kit (Transgen, www . transgen.com), according to the protocol by the manufacturer. The first‐strand cDNA was synthesized with random oligonucleotides using the HiScript® II Reverse Transcriptase system (Vazyme Biotech Co., Ltd). qRT‐PCR was carried out in a total volume of 20 μL containing 2 μL of cDNA, 0.4 μL gene‐specific primers (10 μm ), 10 μL SYBR Green Mix and 7.2 μL of RNase free ddH2O, using the Roche LightCycler480 Real‐time System (Roche, Swiss Confederation). The PCR conditions were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The rice OsActin and 18S rRNA genes were used as internal controls. Primers used for qRT‐PCR are listed in Table S1 . Normalized transcript levels were calculated using the comparative CT method (Livak and Schmittgen, 2001 ). Three biological replications were performed.
3
Wheat EXO70E1-V Ortholog Gene Expression
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Total RNA was extracted by using the Trizol Reagent Kit (Invitrogen, Carlsbad, CA, United States) and analyzed by gel electrophoresis. The first-strand cDNA was synthesized with random oligonucleotides using the HiScript® II Reverse Transcriptase system (Vazyme, Nanjing, China). Quantitative reverse transcription (qRT)-PCR was carried out in a total volume of 20 μL containing 2 μL of cDNA, 0.4 μL of gene-specific primers (10 μm), 10 μL of SYBR Green Mix, and 7.2 μL of RNase free ddH2O, using the Roche LightCycler 480 Real-time System (Roche, Basel, Swiss). The wheat tubulin gene was used as internal controls. The program and data analysis were carried out as described in the method suggested by Wang et al. (2018) (link). Primers used for the qRT-PCR are designed by Primer5 listed in Supplementary Table 1 . Three biological replications were performed. We obtained the in silico expression data of EXO70E1-V ortholog genes in wheat (TraesCS3A02G302600, TraesCS3B02G333800, and TraesCS3D02G299200) induced by Pm from the Triticeae Multi-omics Center wheat gene expression website2 .
4
Quantifying Antioxidant Gene Expression
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Total RNA was extracted using a Trizol Reagent kit (Invitrogen, CA, USA) according to the manufacturers’ instructions [63 (link)]. Three microliters RNA was used in agarose gel electrophoresis to check the quality and integrity of the obtained RNA samples. The first-strand cDNA was synthesized with random oligonucleotides using the HiScript® II Reverse Transcriptase system (Vazyme, Nanjing, China).
The transcription levels of TaCAT (TraesCS4D02G322700), TaAPX (TraesCS2B02G087400), TaSOD (TraesCS7D02G043000) and TaPOD (TraesCS3D02G031800) were analyzed by qRT-PCR. Tubulin was used as the internal control for normalization. Primers used for qRT-PCR were designed by Primer3 (TableS3 ), and three biological replications were performed. qRT-PCR was carried out in a total volume of 20 μL containing 2 μL of cDNA, 0.4 μL gene-specific primers (10 μM), 10 μL SYBR Green Mix, and 7.2 μL RNase free ddH2O, using the Roche LightCycler480 Real-time System (Roche, Basel, Swiss Confederation) [63 (link)]. Finally, the transcription lervel was represented in the form of relative fold change using the 2−ΔΔCT method [76 (link)].
The transcription levels of TaCAT (TraesCS4D02G322700), TaAPX (TraesCS2B02G087400), TaSOD (TraesCS7D02G043000) and TaPOD (TraesCS3D02G031800) were analyzed by qRT-PCR. Tubulin was used as the internal control for normalization. Primers used for qRT-PCR were designed by Primer3 (Table
5
qRT-PCR Analysis of Differential Gene Expression
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Total RNA was extracted using a Trizol Reagent kit (Invitrogen, CA, USA) according to the manufacturer’s instructions and analyzed by gel electrophoresis. The first-strand cDNA was synthesized with random oligonucleotides using the HiScript® II Reverse Transcriptase system (Vazyme, Nanjing, China). qRT-PCR was carried out in a total volume of 20 μL containing 2 μL of cDNA, 0.4 μL gene-specific primers (10 μM), 10 μL SYBR Green Mix and 7.2 μL of RNase free ddH2O, using the Roche LightCycler480 Real-time System (Roche, Basel, Swiss Confederation). The expression was represented in the form of relative fold change using the 2−ΔΔCT method [79 (link)]. Differentially expressed genes between each two samples pair were defined as two-fold up-regulated or two-fold down-regulated genes. Primers used for qRT-PCR are designed by Primer3 (Table S3 ). Three biological replications were performed. Heat map analysis of the expression data was performed using heat map drawing software MeV (version No. 4.7, Institute for Genomic Research, MD, USA)
6
Haplotype-Dependent OsPK5 Expression
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To determine whether two haplotypes affect OsPK5 expression, seeds of ten extreme accessions after 24 h of imbibition (Supplementary Table S2 ) were selected to compare the transcript levels of OsPK5. Dry seeds, germinated seeds, leaf, sheath, panicle, stem, stem node and roots were used to explore the expression pattern of OsPK5 in japonica ‘Nipponbare’ (WT) plants. Total RNA from each plant tissue was isolated using the TransZol Plant reagent kit (TransGen Biotech Co. Ltd., Beijing, China) following the manufacturer’s protocol. First-strand cDNA synthesis was performed with ~500 ng of total RNA using the HiScript® II Reverse Transcriptase system (Vazyme Biotech Co. Ltd. Nanjing, China). qRT–PCR experiments were performed using the Roche LightCycler 480 Real-time System (Roche, Swiss Confederation). The rice ACTIN gene (OsActin, LOC_Os03g50885) and 18S RIBOSOMAL RNA were used as internal references to normalize gene expression using the comparative CT method described by Livak and Schmittgen (2001) (link). The gene-specific primers used for qRT–PCR are listed in Supplementary Table S3 . Three biological replicates were performed.
7
Temporal Expression Profiling of Rice Genes
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Total RNA was extracted from developing grains (at 0, 7, 14, 21, 28, 35 or 42 DAF) and germinating seeds (at 12, 24, 36, 48, 60 and 72 h after imbibition) in Nip using the TransZol Plant kit (Transgen, http://www.transgen.com ) according to the manufacturer's protocol. First‐strand cDNA was synthesized with random oligonucleotides using the HiScript® II Reverse Transcriptase system (Vazyme Biotech Co., Ltd.). qRT‐PCR was carried out in a total volume of 20 μl containing 2 μl of cDNA, 0.4 μl of gene‐specific primers (10 μm ), 10 μl of SYBR Green Mix and 7.2 μl of RNase‐free ddH2O using the Roche LightCycler480 Real‐time System (Roche, Swiss Confederation). PCR conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. Rice OsActin and 18S rRNA genes were used as internal controls. Primers used for qRT‐PCR are listed in Table S4 . Normalized transcript levels of gene expression were calculated using the comparative CT method (Livak and Schmittgen, 2001 ). Transgenic plants carrying the OsHAK21 promoter−GUS fusion construct in Nip were used for a GUS staining assay. Histochemical staining of GUS activity was performed as described by Lagarde et al. (1996 ). Three biological replicates were made.
8
Quantifying mRNA Expression in Liver and Adipose
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Total mRNAs from frozen livers and adipose tissues were extracted using the TRIZOL method, and cDNA was synthesized using the HiScript® II Reverse Transcriptase System (R223-01; Vazyme, Nanjing, China). The real-time quantitative PCR system LightCycler® 480 II (Roche, Hercules, CA, United States) was used for quantitative analysis. All procedures were performed according to the manufacturers’ protocols. The mRNA expression of different genes was normalized to that of β-actin or GAPDH. The sequences of the primers used for qRT-PCR are listed in Supplementary Table 1 .
9
Comprehensive Transcriptomic Analysis of Rice
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Total RNA was extracted from the developing grains (0, 7, 14, 28 and 35 days after flowering; DAF), germinating seeds (0, 6, 12, 24, 30, 36, 48, 54, 60 and 72 h imbibition) and different tissues (root, stem, leaf, sheath, node and spike) of IR26 using the HP Plant RNA kit (Omega, Atlanta), according to the manufacturer's instructions. First‐strand cDNA was synthesized with random oligonucleotides using the HiScriptII Reverse Transcriptase system (Vazyme Biotech Co., Ltd.). The RT‐qPCR was performed on a Roche Light Cycler 480 system using SYBR Green Mix. The Actin gene was used as the internal control for normalization. Primers used for expression analysis are listed in Table S8 . Three biological replicates were made.
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