Soybean tissue samples were lyophilized and stored at –80 °C until use. Total RNA was isolated from soybean tissues using the Trizol reagent according to the supplier’ s instruction. Extracted RNA was treated with DNase to remove contaminating DNA and reverse transcribed using the ReverTran Ace® qPCR RT kit (Toyobo) for reverse transcriptase-PCR. qRT-PCR was performed with an StepOnePlus™ Real-Time PCR System (ABI). PCRs were performed using the SYBR® Green qPCR Master Mixes (Takara) and gene-specific primers (see Supplementary Table S2 ). The PCR conditions consisted of denaturation at 95 °C for 1min, followed by 40 cycles of denaturation at 95 °C for 15s, annealing and extension at 58 °C for 30s. Melt curve analysis was performed on the end products of PCR, to determine the specificity of reactions. Relative quantification of gene expression was calculated according to the ΔΔCt method. The soybean Actin gene (Gm18g52780) was used as internal control.
Revertran ace qpcr rt kit
Manufactured by Toyobo
The ReverTran Ace® qPCR RT kit is a reagent kit designed for reverse transcription and quantitative real-time PCR (qPCR) analysis. It contains the necessary components for the conversion of RNA to complementary DNA (cDNA) and subsequent qPCR amplification.
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7 protocols using revertran ace qpcr rt kit
1
Soybean RNA Isolation and qRT-PCR Analysis
2
Quantitative RT-PCR Analysis of Soybean Gene Expression
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Soybean tissue samples were lyophilized and stored at –80°C until use. Total RNA was isolated from soybean tissues using the Trizol reagent according to the supplier’s instructions. Extracted RNA was treated with DNase to remove contaminating DNA and reverse transcribed using the ReverTran Ace® qPCR RT kit (Toyobo) for reverse transcriptase-PCR. qRT-PCR was performed with a StepOnePlusTM Real-Time PCR System (ABI). PCRs were performed using the SYBR® Green qPCR Master Mixes (Takara) and gene-specific primers listed in Supplementary Table S2 . The PCR conditions consisted of denaturation at 95°C for 1min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 58°C for 30 s. Relative gene expression was calculated as previously described (Livak and Schmittgen, 2001 (link)). The soybean actin gene (Glyma18g52780, 5′-GTGCACAATTGATGGACCAG-3′ and 5′-GCACCACCGGAGAGAAAATA-3′) was used as an internal control.
3
Soybean Gene Expression Analysis by qRT-PCR
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Soybean tissue samples were lyophilized and stored at −80 °C until use. Total RNA was isolated from soybean tissues using the Trizol reagent according to the supplier’s instruction. Extracted RNA was treated with DNase to remove contaminating DNA and reverse transcribed using the ReverTran Ace® qPCR RT kit (Toyobo) for reverse transcriptase-PCR. qRT-PCR was performed with an StepOnePlus™ Real-Time PCR System (ABI). PCRs were performed using the SYBR® Green qPCR Master Mixes (Takara) and gene-specific primers (Supplemental Table 2 ). The PCR conditions consisted of denaturation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 5s, 55 °C annealing for 30s and extension at 72 °C for 30s. Melt curve analysis was performed on the end products of PCR, to determine the specificity of reactions. Relative quantification of gene expression was calculated according to the ΔΔCt method. The soybean actin gene (Gm18g52780) was used as internal control. The heatmap was visualized using Heatmapper Plus tool at the Bio-Array Resource for Plant Functional Genomics.
4
Quantitative Real-Time PCR Analysis of Gene Expression
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Arabidopsis and soybean samples were lyophilized and stored at −80 °C until use. Total RNA was isolated from plant tissues using the Trizol reagent according to the supplier’s instruction. Extracted RNA was treated with DNase to remove contaminating DNA and reverse transcribed using the ReverTran Ace® qPCR RT kit (Toyobo) for reverse transcriptase-PCR. qRT-PCR was performed with an StepOnePlus™ Real-Time PCR System (ABI). PCRs were performed using the SYBR® Green qPCR Master Mixes (Takara) and gene-specific primers listed in Supplemental Table 3 . Relative gene expression was calculated as previously described50 (link). Soybean Actin gene (Glyma18 g52780, 5′-GTGCACAATTGATGGACCAG-3′ and 5′-GCACCACCGGAGAGAAAATA-3′) was used as internal control.
5
Quantifying Soybean Gene Expression by qRT-PCR
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Gene expression analysis using qRT-PCR was performed as previously described53 . Soybean samples were lyophilized and stored at −80 °C until use. Total RNA was isolated from plant tissues using the Trizol reagent according to the supplier’s instruction. Extracted RNA was treated with DNase to remove contaminating DNA and reverse transcribed using the ReverTran Ace® qPCR RT kit (Toyobo) for reverse transcriptase-PCR. qRT-PCR was performed with an StepOnePlus™ Real-Time PCR System (ABI). PCRs were performed using the SYBR® Green qPCR Master Mixes (Takara) and gene-specific primers (Supplemental Table 2 ). Relative gene expression was calculated as previously described54 (link). Soybean Actin gene (Glyma18g52780, 5′-GTGCACAATTGATGGACCAG-3′ and 5′-GCACCACCGGAGAGAAAATA-3′) was used as internal control.
6
Validating RNA-seq Data by qRT-PCR
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To validate the RNA-Seq data, the transcript levels of selected up- or down-regulated genes were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Specific primers were designed in the website (http://primer3.ut.ee/ ) (Additional file 16 : Table S6). RNA was reverse transcribed using the ReverTran Ace® qPCR RT kit (Toyobo). qRT-PCR was performed on an LightCycler 480 II (Roche, Switzerland) using the SYBR® Green qPCR Master Mixes (Takara, Japan). The PCR conditions consisted of denaturation at 95 °C for 30s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 30 s. Melt curve analysis was performed to determine the specificity of reactions. The relative expression levels were calculated according to the ΔΔCt method. The eggplant Actin gene (Sme2.5_01462.1_g00018.1) was used as the internal control.
7
Quantitative Analysis of Gene Expression
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The quantitative reverse-transcription PCR (qRT-PCR) was applied to analyze the expression levels of 16 DEGs among the five varieties. First-strand cDNA was syntheized by the ReverTran Ace qPCR RT kit (Toyobo). The primers used for qRT-PCR were designed by Primer 3 (http://primer3.ut.ee , accessed on 12 June 2021). All primers are listed in Supplementary Table S3 . qRT-PCR was performed as previously described [6 (link)]. The gene expression levels were normalized with SmGAPDH as an internal control and were calculated using the 2−ΔΔCt method [49 (link)].
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