The expression patterns of all genes were analyzed via qRT-PCR. The cDNA of all samples were prepared using PrimeScript™ RT Master Mix (Takara, Shiga, Japan). The expression levels of four artemisinin biosynthetic genes were analyzed in the all different samples before transcriptome sequencing. Eight TFs (c119965_g1, c117361_g1, c122024_g1, c95172_g1, c64067_g1, c117542_g1, c109401_g1, c113821_g1) were selected for verification of the sequencing and computational results from four sequenced samples after transcriptome sequencing. All reactions were carried out in 96-well plates on the Roche LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) and used the SYBR Green qPCR Master Mix (Tiangen, Beijing, China) according to the manufacturer's instructions with three replicates. The cycling conditions used for qRT-PCR were: 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 20 s and extension at 72°C for 20 s. The relative expression levels of the genes were normalized to the internal control gene A. annua β-Actin, and determined by the relative 2−ΔΔCt method. All primers used in qRT-PCR are listed in Table S1 .
Sybr green qpcr master mix
Manufactured by Tiangen Biotech
Sourced in China
SYBR Green qPCR Master Mix is a ready-to-use solution for quantitative PCR analysis. It contains SYBR Green I dye, DNA polymerase, and necessary reagents for PCR amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rnaprep pure tissue kit, by Tiangen Biotech (3 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Abi 7300 pcr machine, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Lightcycler 96 real time pcr system, by Roche (1 mentions)
First strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Prism 6, by GraphPad (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Trizol, by Tiangen Biotech (1 mentions)
First strand cdna synthesis kit, by Transgene (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Reverse transcriptase, by Promega (1 mentions)
Qtower real time pcr system, by Analytik Jena (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Superscript tm 2 first strand synthesis kit, by Takara Bio (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
19 protocols using sybr green qpcr master mix
1
Quantifying Artemisinin Pathway Genes
2
Quantifying SERCA2a and SUMO1 mRNA
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Total RNA were extracted from cultured cells using TRIzol. The concentration of total RNA was determined by spectrophotometry. Purity of RNA was confirmed by an optical density 260/280 nm ratio between 1.8 and 2.0. Total RNA was reverse-transcribed to cDNA at 70 °C for 5 min, and cooled on ice. A first-strand cDNA synthesis kit (Tiangen, Beijing, China) was used at 42 °C for 50 min, followed by a step of 95 °C for 5 min to terminate the reaction.
Real time PCR analysis was carried out using SYBR Green qPCR Master mix (Tiangen, Beijing, China), the reaction was run in a total volume of 20 μl containing 1 μl cDNA as a template. Thermal cycling was by the methods of Wu50 (link). The fluorescence of the SYBR green dye was plotted as a function of PCR cycle number. To confirm the specificity of amplification, the PCR products from each primer pair were analyzed by their melting curves. The Δ CT values (Ct = cycle threshold value) of the target genes (SERCA2a and SUMO1) were calculated by subtracting the values of the experimental group from the housekeeping gene (GAPDH) values. The 2-∆ (∆Ct) method was used to calculate the relative expression of SERCA2a and SUMO1 mRNA. The primer sequences designed with online tools were as follows:
Real time PCR analysis was carried out using SYBR Green qPCR Master mix (Tiangen, Beijing, China), the reaction was run in a total volume of 20 μl containing 1 μl cDNA as a template. Thermal cycling was by the methods of Wu50 (link). The fluorescence of the SYBR green dye was plotted as a function of PCR cycle number. To confirm the specificity of amplification, the PCR products from each primer pair were analyzed by their melting curves. The Δ CT values (Ct = cycle threshold value) of the target genes (SERCA2a and SUMO1) were calculated by subtracting the values of the experimental group from the housekeeping gene (GAPDH) values. The 2-∆ (∆Ct) method was used to calculate the relative expression of SERCA2a and SUMO1 mRNA. The primer sequences designed with online tools were as follows:
3
Validating RNA-seq Data with qRT-PCR
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In order to verify the accuracy of the RNA-seq, a qRT-PCR analysis was used to assess the quality of the RNA-seq data. Total RNA was extracted from root tissue with TRIzol according to the manufacturer’s instructions (TIANGEN, China), and the isolated RNA was reverse-transcribed into cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, China). The expression of randomly selected genes was monitored by qRT-PCR using the SYBR Green qPCR Mastermix (TIANGEN, China) real-time PCR system by Bio-Rad CFX 96™ following the manufacturer’s instructions. Detailed information about the primer sequences for qRT-PCR was provided by the primer3 platform (http://frodo.wi.mit.edu/primer3/ ; Table S6 ). The qRT-PCR was used on three biological replicates with the expression levels of the genes determined using the 2−−ΔΔCT method. Expression levels were normalized against the GAPDH. Data are represented as mean values ± standard deviation, and the GraphPad Prism 6 software was employed to draw the histogram.
4
Quantitative Analysis of mRNA and miRNA
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Total RNA was isolated from frozen samples using the RNAiso Plus Kit (Takara, Dalian, China). First-strand complementary cDNA synthesis for gene quantification was performed with the First-Strand cDNA Synthesis kit (TransGen, Beijing, China) using oligo(dT)18 primers. For miRNA quantification, first-strand complementary cDNA synthesis was performed, using miRNA-specific primers. The qRT-PCR and stem-loop qRT-PCR were performed with SYBR green qPCR Master Mix (Tiangen, Beijing, China) for gene and miRNA quantification, respectively [70 (link)]. Amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). All reactions were performed in triplicate. After PCR, the data were computed by the comparative Ct method (2−∆∆Ct method). Actin and soybean miR1520d were used as reference genes for gene and miRNA normalization, respectively. The primer sequences are listed in the Table S5 .
5
Quantifying miR-31 and RhoBTB1 Expression
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Total RNA was extracted from transfected A-431 cells using TRIzol reagent (Invitrogen, ThermoFisher Scientific, Inc.) and then reverse-transcribed into cDNA. RT-qPCR was performed using the SYBR Green qPCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China) on an ABI 7300 PCR machine (Applied Biosystems, Inc., Foster, CA, USA). The sequences of the primers used to detect miR-31 and U6 were as follows: miR-31, forward 5′-GGAGAGGCAAGATGCTGGCA-3′; U6, forward 5′-CGCAAGGATGACACGCAAATTC-3′; and a universal downstream reverse primer, 5′-GTGCAGGGTCCGAGGT-3′. The primers used for detection of RhoBTB1 were as follows: forward 5′-GGAGTGAAGGAGCCTGTGAG-3′; and reverse 5′-TGCCAATGAACCCCTTACTC-3′. qPCR cycling conditions were as follows: 95°C for 10 min, and then 95°C for 15 sec and 50°C for 2 min, for 40 cycles, followed by 60°C for 1 min. The melting curve was 65–95°C. The relative mRNA expression levels were calculated as 2−∆∆Cq and were normalized against U6.
6
Quantitative Expression Analysis of ABC Transporters
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Total RNA was extracted from cells using the TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) chloroform extraction method. The cDNA was prepared from 1 µg total RNA using reverse transcriptase (Promega Corporation, Madison, WI, USA). Total cellular RNA was quantified by qPCR analysis using gene-specific primer pairs. RT-qPCR was performed by the SYBR-Green qPCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China) on an ABI 7300 PCR machine (Applied Biosystems, Inc., Foster, CA, USA). qPCR cycling conditions were as follows: 95°C for 5 min, and then 95°C for 15 sec, 65°C for 15 sec and 72°C for 32 sec, for 40 cycles. The melting curve was 60–95°C. The relative mRNA expression levels were calculated as 2−∆∆Cq method (18 (link)). Oligonucleotides used for the analysis were as follows: ABCB1 forward, 5′-CTAGAAGGTTCTGGGAAGAT and reverse, 5′-GAGTTTCTGTATGGTACCTG; ABCC1 forward, 5′-GGCTCAAGGAGTATTCAGAG and reverse, 5′-CCATCGATGATGATCTCTCC; EIF4G forward, 5′-CTCTGAGACCGTTGGGCAAA and reverse, 5′-GCGACGAATGCACCAATGT; ABCG2 forward, 5′-GCAGGTCAGAGTGTGGTTTC and reverse, 5′-GACAGCCAAGATGCAATGGT; and 18S ribosomal RNA forward, 5′-CCTGGATACCGCAGCTAGGA and reverse, 5′-GCGGCGCAATACGAATGCCCC. Each reaction was performed in triplicate and three independent experiments were run.
7
qRT-PCR Gene Expression Analysis
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Eight genes were performed using qRT-PCR and Actin as an internal reference to verify the RNA-seq results. First-strand complementary cDNA synthesis for gene quantification was carried out using the First-Strand cDNA synthesis Kit (TranGen, Beijing, China). Gene expression was quantified by SYBR green qPCR Master Mix (Tiangen, Beijing, China) according to the manufacturer’s instructions. Three biological and three technical replicates were performed for each transgenic line, and the relative expression levels were calculated using the 2–ΔΔCt method. The primer sequences are listed in the Supplementary Table S2 .
8
Quantifying Jasmonic Acid-Responsive Genes
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The expression levels of selected JA-responsive genes (i.e. LoxD, MYC2, JAZ1, and RBOH) were analyzed using SYBR Green-based RT-qPCR on a qTOWER real-time PCR system (Analytik Jena, Jena, Germany). PCR assays were performed in a 20 μL reaction mixture containing 10 μL of SYBR Green qPCR Master Mix (Tiangen Biotech, Beijing, China), 8.8 μL of sterile water, 0.6 μL of cDNA, and 0.3 μL each of forward and reverse primers. PCR protocol was 10 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 58 °C, and 40 s at 72 °C. The actin gene was used as the reference gene. Primers used in this study are listed in Supplemental Table S1 . Sterile water instead of cDNA served as the negative control. All amplifications were performed in triplicate. The relative expressions of these genes were calculated with the 2−ΔΔCT method (Livak and Schmittgen 2001 (link)).
9
Quantifying Gene Expression in Skin Samples
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Skin samples were treated with the TRIzol reagent (9109, Takara, Japan). Then, total RNA was extracted in accordance with the manufacturer’s instructions. Next, RNA was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (RR036A, Takara, Tokyo). qRT-PCR was carried out using the specific primers (Supplementary Table 1
10
qPCR Analysis of miRNA-29c Expression
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A real-time quantitative PCR was used to measure the relative expression level of miRNA-29c in the samples of DM and control subjects. To be specific, real-time quantitative PCR was carried out using the reagents and protocol of the SYBR Green qPCR Master Mix (FP411, TIANGEN Inc., Beijing, P.R. China). MiRNA-29c and U6 primer sequences were purchased from TIANGEN company (CD201-0332, CD201-0145, TIANGEN Inc., Beijing, P.R. China). Briefly, in the first step, the template was denatured at 95 °C for 5 min. The second step consisted of 45 cycles at 94 °C for 20 s and 60 °C for 34 s. Each sample was run in triplicate. In this study, the 2−ΔΔCt method was applied to quantify. U6 was used as a loading control to normalize the expression level of the target protein.
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