cDNA microarray analysis was used to examine differential gene expression between 0% and 1% KRG extract group. Total RNA was isolated from 6 tissues randomly selected in each group using the Trizol extraction reagent (Invitrogen, Carlsbad, CA). Digital Genomics Inc. (Seoul, Korea) performed duplicate examinations on GenePlorerTMtwin chipTM-Rat 5K containing 4,863 gene probes with mixed total RNA. Global median, intensity/location-dependent normalization was performed to analyze all data. Genes were considered differentially expressed when the log2 ratio was more than 1 or less than -1. To identify the pathway altered by the 1% KRG extract, differentially expressed gene data underwent further analyses using KEGG pathway. A DAVID (the database for annotation, visualization and integrated discovery) bioinformatics resources (http://david.abcc.ncifcrf.gov/ ) was used to perform a functional analysis for those genes 31 (link).
Trizol extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
TRIzol is a reagent used for the isolation of total RNA from various biological samples, including cells, tissues, and bodily fluids. It is based on the single-step RNA isolation method developed by Chomczynski and Sacchi. TRIzol utilizes a mixture of phenol and guanidine isothiocyanate to effectively lyse cells and denature protein complexes, allowing for the separation and purification of RNA.
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Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Primer express version 3, by Thermo Fisher Scientific (2 mentions)
Sybr green premix ex taq, by Takara Bio (2 mentions)
Cfx384 real time system, by Bio-Rad (2 mentions)
Rnase free dnase kit, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr kit, by Takara Bio (2 mentions)
Sybr green qpcr supermix, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Verso cdna kit, by Thermo Fisher Scientific (1 mentions)
7500 fast real time polymerase chain reaction system, by Thermo Fisher Scientific (1 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Primescript rt pcr reagent kit, by Takara Bio (1 mentions)
Abi 7300, by Thermo Fisher Scientific (1 mentions)
Rotor gene qpcr system, by Qiagen (1 mentions)
Revertaid rt kit, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
42 protocols using trizol extraction reagent
1
cDNA Microarray Analysis of KRG Extract Effects
2
Quantification of mRNA and miRNA Expression
3
Quantifying Terpenoid Gene Expression in Berries
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A total of 50–100 mg of frozen berry samples representing the different development stages was ground in a mortar and the resultant powder was used for the RNA extraction with the TRIzol extraction reagent (Invitrogen, Carlsbad, CA, USA). Agarose gel electrophoresis was used to assess the quality of every RNA sample and high-quality RNA samples with an A260/A280 ratio of 1.8:2.1 were used as the template material for the qRT-PCR analyses. Specifically, complementary DNA (cDNA) was transcribed using the Takara Prime Script TM-RT PCR reagent Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. Since the monoterpene volatiles showed the most significant variations after the elicitor treatment, we quantified the expression of genes in the terpenoid metabolic pathways by qRT-PCR analysis (ABI7300, Applied Biosystems, Milan, Italy). Relative transcript levels were calculated using the 2−ΔΔCt method [53 (link)] and VvActin was used as the reference for normalization purposes. All reactions, including non-template controls, were performed in triplicate. Sequences of the primers used to amplify the VvDXS1, VvDXS3, VvDXR and VvHDR genes were referenced from Martin [43 (link)]; VvPNGer and VvPNlinNer1 were referenced from Matarese [47 (link)].
4
Quantitative Analysis of Antioxidant Genes
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Total RNA was isolated from root tissues using TRIZOL extraction reagent (Invitrogen, Gaithersburg, MD, USA). RNA purity was verified based on the ratio (>1.9) of 260/280 nm absorbance. DNA-free total RNA (5 μg) from different treatments was used for first-strand cDNA synthesis in a 20 μL reaction volume (Thermo Scientific, MD, Lithuania) according to the manufacturer’s instructions. Real-time quantitative PCR reactions were performed using a Mastercycler® ep realplex real-time PCR system (ABI7500, MD, USA) with Bestar® SybrGreen qPCR mastermix (DBI, Bioscience Inc., Germany) in a 20 μL reaction volume according to the user manual.
PCR primers targeting GCS, GS, GR1, GR2, GPX and GST were designed using Primer Express®version3.0 (Applied Biosystems), and actin was designed following Xiao et al [44 ]. All primers (S1 Table ) were synthesized by Genewiz Bio-engineering Ltd. Company (Suzhou, China). The expression levels are presented relative to those of corresponding control samples at the indicated time after normalization to actin transcript levels.
PCR primers targeting GCS, GS, GR1, GR2, GPX and GST were designed using Primer Express®version3.0 (Applied Biosystems), and actin was designed following Xiao et al [44 ]. All primers (
5
Quantitative Real-Time PCR Analysis
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Total RNA was extracted by using TRIzol extraction reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was performed using the Revert Aid RT Kit, in accordance with manufacturer’s instruction (Thermo Scientific, Waltham, USA. qRT-PCR assays were carried out in Rotor-Gene®qPCR System (Qiagen, Hilden, Germany), using SsoAdvanced Universal SYBR Green Supermix (Biorad, Hercules, CA, USA). Briefly, each reaction was performed in a final volume of 10 μL containing 1 μL of the cDNA, 1 μL of forward and 1 μL of reverse primers, 5 μL of SsoAdvanced Universal SYBR Green Supermix and 1 μL of nuclease-free water. Primers were designed based on the mouse GenBank sequences for IL-6, IL-1β, and TNFa, and are reported in Table 6 . The amplification protocol was based on an initial heat activation at 95 °C for 30 s, followed by 35 cycles of denaturation at 95 °C for 15 s and combined annealing/extension at 60 °C for 30 s. The relative expression of mRNA was normalized by β-Actin and calculated by the 2−ΔΔCt method.
6
RNA Extraction and RT-PCR Analysis
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The RNA was extracted using TRIzol extraction reagent (Invitrogen, 15596018). The complementary DNA was synthesized using PrimeScript™ RT reagent Kit (Takara, RR047A). RT-PCR was performed using SYBR Green Premix Ex Taq (Takara, AK8806) in a CFX384 Real-Time Systems (Bio-Rad, C1000, Touch). Sequences of the primers used are shown in Supplementary Table 1 .
7
Comprehensive Gene Expression Analysis
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Total RNA was isolated using the TRIzol extraction reagent (Invitrogen) or an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized with M-MLV Reverse Transcript reagent (Invitrogen). Quantitative real-time PCR was performed with SYBR Green PCR kit (TaKaRa Bio Inc, Otsu, Shiga, Japan) and analyzed in an ABI 7500 Sequence Detection System. The expression level of each gene was normalized to the expression level of GAPDH. PCR primers were listed in Supplementary Table S1 .
8
Quantifying Gene Expression in Cells
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Total RNA was extracted from cells by TRIzol extraction reagent (Invitrogen) following the manufacturer’s instructions (n = 4). Expression levels of numerous genes were determined by PCR and qPCR. Angiogenesis-related genes included CD34, PECAM1, Flk1, and VE-cadherin. Stemness-related genes included c-Myc, Klf4, and CD271. NO biosynthesis–associated genes included intercellular adhesion molecule 1 (ICAM1), interleukin-6 (IL-6), clusterin (CLU), pentraxin-related protein 3 (PTX3), and prostaglandin-I synthase (PTGIS). Complementary DNA (cDNA) was synthesized using the AccuPower CycleScript RT PreMix (Bioneer) according to the manufacturer’s instructions. PCR was performed on a T100 Thermal Cycler (Bio-Rad) using a HiPi Plus 5× PCR premix (Elpisbio), and the products were analyzed by 2% (w/v) agarose gel electrophoresis. Real-time qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using SYBR Green PCR mix (Thermo Fisher Scientific). Each gene expression was normalized to 40S ribosomal protein S18 (RPS18) and analyzed by the relative quantification 2−ΔΔCt method. The values were further normalized to the corresponding samples in the TCPS culture and are presented as fold changes. Primer sequences are listed in table S4.
9
Radish Transcriptional Regulation of Flavonoid Biosynthesis
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Total RNA was extracted from radish hypocotyl samples using Trizol extraction reagent (Invitrogen, Gaithersburg, MD, USA) and high purity of RNA with ratio of 260/280 nm > 1.9 was used. First-strand cDNA was synthesized in a 20 μL reaction volume (Thermo Scientific, MD, Lithuania) containing 1 μL of RevertAid M-MuLV reverse transcriptase and 1 μL of oligo (dT)18 primer according to the manufacturer’s instructions. A Mastercycler® ep realplex real-time PCR system (ABI7500, MD, USA) with Bestar® SybrGreen qPCR mastermix (DBI, Bioscience Inc., Germany) in a 20 μL reaction volume was used to perform the real-time quantitative PCR reactions according to user manual.
Primer Expressversion 3.0 (Applied Biosystems) was used to design all PCR primers targeting actin, PAL, CHS, CHI, F3H, DFR, LDOX, ANS, and UFGT (Su et al., 2014 (link)). All primers (Supplementary Table1 ) were synthesized by Genewiz Bio-engineering Ltd. Company (Suzhou, China). The identification of PAL, F3H, ANS and UFGT in radish genes was based on using their Arabidopsis orthologs for homology search in databank of R. sativus available at http://bioinfo.bti.cornell.edu/radish (Shen et al., 2013 (link)). The transcription levels were presented as values compared to those of corresponding control samples, after normalization to actin expression levels.
Primer Expressversion 3.0 (Applied Biosystems) was used to design all PCR primers targeting actin, PAL, CHS, CHI, F3H, DFR, LDOX, ANS, and UFGT (Su et al., 2014 (link)). All primers (Supplementary Table
10
Total RNA Extraction from Fungal Samples
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Total RNA was extracted from a total of 12 samples (3 replicates per treatment) made up by 100 mg of fungal hyphae and spore combined using TRIzol extraction reagent (Invitrogen Life Technologies, USA) according to the manufacturer’s protocol. Then, fungal DNA was removed from extractions with DNAseI followed by the removal of rRNA using Ambion’s Poly (A) Purist kit. The integrity of the RNA was detected by agarose gel electrophoresis and the concentration was determined using Nano Drop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The RNA integrity was further evaluated using an Agilent Technology 2100 Bioanalyzer (Agilent Technologies, USA).
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