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.
Sybr green qpcr master mixes
Manufactured by Takara Bio
Sourced in China
SYBR® Green qPCR Master Mixes are ready-to-use solutions for quantitative real-time PCR (qPCR) analysis. The mixes contain SYBR® Green I dye, which binds to double-stranded DNA and emits fluorescence upon binding. This allows for the detection and quantification of DNA targets during the qPCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Revertran ace qpcr rt kit, by Toyobo (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Stem loop rt primer, by RiboBio (1 mentions)
Viia 7 dx system, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Abi 7300 system, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
10 protocols using sybr green qpcr master mixes
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
Quantifying miRNA and mRNA Levels
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Total RNA was isolated using TRIzol reagent (Invitrogen, USA). cDNA was synthesized by Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The RT reaction for miR-150 was carried out with stem-loop RT primers (RiboBio, China). Quantitative PCR was performed using SYBR Green qPCR Master Mixes (Takara, China). Relative expression was determined using U6 (primers from RiboBio, China) as the internal control for miRNAs and GAPDH as the internal control for mRNAs of other genes (primers listed in Table 1 and Supplementary Table S1 ).
4
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.
5
Quantification of Iron Regulatory Genes
6
Quantitative Analysis of Gene Expression
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Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian, Liaoning, China). Reverse transcription (1 μg of total RNA) was performed with the PrimeScript RT reagent kit (TaKaRa, Dalian, Liaoning, China). The cDNA was subjected to quantitative PCR (qPCR) on a ViiA 7 Dx system (Applied Biosystems, Foster, CA, United States) using SYBR green qPCR master mixes (TaKaRa, Dalian, Liaoning, China). The expression levels of target genes were normalized to that of the internal control gene (rpoD) using the 2–ΔΔCt method. More details about primers are listed in Supplementary Table 3 .
7
Quantitative Analysis of Glioma RNA
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Trizol (Invitrogen) was used to extract miRNAs and total RNA from glioma tissues and cell lines according to the manufacturer’s protocol. DNase I (Roche, Indianapolis, IN, USA) was used to remove residual DNA. QRT-PCR was performed to detect the expressions of RHPN1-AS1, miR-625-5p and REG3A. A cDNA synthesis kit (TaKaRa Biotechnology Co., Ltd, Dalian, China) and oligo (dT) primers were applied to transcribe RNA (2 μg) to cDNA, and SYBR Green qPCR Master Mixes (TaKaRa) was then used for following qRT-PCR according to the manufacturer’s instruction. GAPDH was used for normalization. The oligonucleotides, used as PCR primers, were as follows: lncRNA RHPN1-AS1 5ʹ-CTAGCCAGGAGGTTTCGC-3ʹ and 5ʹ-TCCGCAACAAGCACACA-3ʹ, GAPDH 5ʹ-CACCCACTCCTCCACCTTTG-3ʹ and 5ʹ- CCACCACCCTGTTGCTGTAG-3ʹ, miR-625-5p 125ʹ-AGCGCGACTATAGAACTTTCC-3ʹ and 5ʹ-CTCCTACTCCCTCCCTCATC-3ʹ, REG3A 5ʹ-GCCTGTGTTTGGTGTGC-3ʹ and 5ʹ-ATGAGGTGGTCAGGTTGG-3ʹ; the ABI 7300 system (Applied Biosystem, Foster City, CA, USA) was performed to initially incubating the samples at 95°C for 10 mins, and then at 95°C for 10 mins, followed by 40 cycles of incubation at 95°C for 15 s and 60°C for 45 s. The 2−ΔΔCt method was employed to calculate the fold changes. All data represent the average of three replicates.
8
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.
9
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.
10
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.
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