Total cellular RNA was extracted from hBMMSCs of P7 and P65 using the TRIzol reagent as instructed by the manufacturer (Invitrogen Life Technologies). RNA (1 μg; RIN value >6) was used for generation of second-strand cDNA, and cRNA was amplified with the Oligo dT primer, then biotinylated and fragmented with One-Cycle Target Labeling and control reagents (Affymetrix, Inc., Santa Clara, CA, USA), followed by hybridization to U133 Plus 2.0 array overnight (17 h) according to the manufacturer’s instructions. Finally, the hybridized DNA microarray was fluorescence stained with GeneChipFluidics Station 450 (Affymetrics) and scanned with a GeneChip Scanner 3000 (Affymetrics). GCOS1.2 Affymetrix GeneChip Command Console (TGCC) was employed to extract the data in the microarray followed by analysis with the Robust Multi-Array Average (RMA) method. The relative gene expression was calculated. A difference of >2 or <0.5-fold and reliability (P<0.05) was considered to indicate significant differences in gene expression.
One cycle target labeling and control reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
The One-Cycle Target Labeling and Control Reagents are a set of reagents for the preparation of labeled cRNA targets from total RNA samples for use in gene expression analysis using microarray technologies. The reagents include components necessary for first-strand cDNA synthesis, second-strand cDNA synthesis, in vitro transcription, and cRNA purification.
Automatically generated - may contain errors
Lab products found in correlation
Total rna mouse gene 1.0 st array, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Oligo dt primer, by Thermo Fisher Scientific (2 mentions)
Affymetrix scanner 3000, by Thermo Fisher Scientific (2 mentions)
Genechip command console software, by Thermo Fisher Scientific (2 mentions)
Rnasey mini kit, by Qiagen (2 mentions)
Genespring software, by Agilent Technologies (2 mentions)
Nanodrop nd 2100, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
U133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mirvana rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Mouse gene 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna, by Takara Bio (1 mentions)
Mirvanatm rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Rat genome 230 2.0 array, by Thermo Fisher Scientific (1 mentions)
Genechip rat genome 230 2.0 array, by Thermo Fisher Scientific (1 mentions)
Trizol ls, by Thermo Fisher Scientific (1 mentions)
10 protocols using one cycle target labeling and control reagents
1
Transcriptome Analysis of Bone Marrow Mesenchymal Stem Cells
2
Transcriptome Profiling of Tumor and Normal Tissues
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Total RNA was isolated from 20 samples (collected from Henan Provincial People’s Hospital), including ten tumor tissues and ten adjacent normal tissues, using an mirVana™ RNA isolation kit (AM1561, Thermo Fisher, USA). RNA clean-up was performed using RNasey Mini Kit (Qiagen p/n 74,104). Quantification and quality control were achieved by NanoDrop ND-2100 (ThermoFisher) and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). cDNA synthesis and biotin labeling of cRNA were performed using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, USA) and the GeneChip™ Hybridization, Wash and Stain Kit (Affymetrix) following the manufacturer’s protocol. The purified and fragmented labeled cRNAs were hybridized onto the Affymetrix PrimeView™ Human Gene Expression microarray. After washing and staining, the arrays were scanned and analyzed using Affymetrix Scanner 3000 (Affymetrix). GeneChip Command Console Software (version 4.0, Affymetrix) and Genespring software (version 14.9, Agilent Technologies) were used to analyze the transcriptome data. Differentially expressed genes (DEGs) from TCGA were analyzed using DESeq2 package within R language (version 3.4.3).
3
RNA Pooling and Microarray Analysis in Mouse Model
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Equal quantities of RNA from mice of the same genotype and time point were pooled for microarray assays. For sham-infected mice, equal quantities of RNA from mice of the same genotype and all three time points were pooled as control uninfected mice. Samples were labeled for GeneChip analysis using the One-Cycle Target Labeling and Control Reagents (Affymetrix). The gene expression array used was the Affymetrix Mouse Gene 1.0 ST array. All steps of target labeling, hybridization, and scanning were performed according to the manufacturer's protocol. The entire microarray dataset is available in Supplementary Table 1 .
4
Hippocampal Gene Expression Analysis
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Each mouse was used at two months of age. An RNeasy Mini Kit (74104, Qiagen, Valencia, CA, USA) was used for extraction of total RNA from the hippocampus. To synthesize biotinylated cRNA, total RNA was processed using one-cycle target labeling and control reagents (Affymetrix, Santa Clara, CA, USA), and hybridized to a Total RNA Mouse Gene 1.0 ST Array (Affymetrix) with three biological repeats per group. Raw data that were parametrically normalized [39 (link)] were statistically tested by two-way ANOVA [40 ] at p < 0.001.
5
Transcriptomic Analysis of Theanine Ingestion in Mice
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The mice were housed confrontationally for three days after single housing for one month. Group-housed mice were kept in a group of six for one month. Mice were anesthetized with isoflurane and blood was removed from the jugular vein. The hippocampus was removed and frozen immediately. Total RNA was extracted from the hippocampus using an RNeasy Mini Kit (74104, Qiagen, Valencia, CA, USA). Total RNA was processed to synthesize biotinylated cRNA using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA, USA) and then hybridized to a Total RNA Mouse Gene 1.0 ST Array (Affymetrix), with three biological repeats per group. Raw data were parametrically normalized [45 (link)] by using the SuperNORM data service (Skylight Biotech Inc., Akita, Japan). The significance of theanine ingestion was statistically tested by two-way ANOVA [46 (link)] at p < 0.001.
To compare the effects of theanine ingestion in the control under group or confrontational housing, we performed principal component analysis (PCA) [47 (link)] on ANOVA-positive genes [48 (link)]. To reduce the effects of individual variability among samples, the axes of PCA were estimated on a matrix of each group’s sample means and applied to all data, which were centered using the sample means of control mice under group housing.
To compare the effects of theanine ingestion in the control under group or confrontational housing, we performed principal component analysis (PCA) [47 (link)] on ANOVA-positive genes [48 (link)]. To reduce the effects of individual variability among samples, the axes of PCA were estimated on a matrix of each group’s sample means and applied to all data, which were centered using the sample means of control mice under group housing.
6
Green Tea Catechin Effects on Mouse Hippocampus
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The mice were housed in groups of 6 for 1 month and ingested water containing green tea catechin or nothing (control). Every three mice 2 months of age were anesthetized with isoflurane and blood was removed from the jugular vein. The hippocampus was removed and frozen immediately. Total RNA was extracted from the hippocampus using an RNeasy Mini Kit (74104, Qiagen, Valencia, CA, USA). Total RNA was processed to synthesize biotinylated cRNA using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA, USA) and then hybridized to a Total RNA Mouse Gene 1.0 ST Array (Affymetrix), with 3 biological repeats per group. Raw data were parametrically normalized [50 (link)] by using the SuperNORM data service (Skylight Biotech Inc., Akita, Japan). The significance of GT-catechin ingestion was statistically tested by two-way ANOVA [51 (link)] at p < 0.001.
To compare the effects of GT-catechin ingestion, we performed principal component analysis (PCA) [52 (link)] on ANOVA-positive genes [53 (link)]. To reduce the effects of individual variability among samples, the axes of PCA were estimated on a matrix of each group’s sample means and applied to all data, which were centered using the sample means of control mice.
To compare the effects of GT-catechin ingestion, we performed principal component analysis (PCA) [52 (link)] on ANOVA-positive genes [53 (link)]. To reduce the effects of individual variability among samples, the axes of PCA were estimated on a matrix of each group’s sample means and applied to all data, which were centered using the sample means of control mice.
7
Theanine Modulates Hippocampal Transcriptome
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The mice housed in groups of 6 for 1 month were fed water containing theanine or nothing (control). The hippocampus was removed from each mouse and frozen immediately. Total RNA was obtained from the hippocampus using a purification kit (NucleoSpin® RNA, 740955, TaKaRa Bio Inc., Shiga, Japan). Biotinylated cRNA was synthesized from this total RNA using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA, USA) and hybridized to Total RNA Mouse Gene 1.0 ST Array (Affymetrix). Three biological replicates were performed for each group. The raw data were normalized using the SuperNORM data service (Skylight Biotech Inc., Akita, Japan) [35 (link)]. The significance of theanine ingestion was tested by two-way ANOVA at p < 0.001 [36 (link)]. To compare the effects of theanine intake, principal component analysis (PCA) was performed on ANOVA-positive genes [37 (link),38 (link)].
8
Transcriptome Analysis of Tumor and Normal Tissues
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Total RNA was isolated from 20 samples, including ten tumor tissues and ten adjacent normal tissues, using an mirVana TM RNA isolation kit (AM1561, Thermo Fisher, USA). RNA clean-up was performed using RNasey Mini Kit (Qiagen p/n 74104). Quanti cation and quality control were achieved by NanoDrop ND-2100 (ThermoFisher) and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). cDNA synthesis and biotin labeling of cRNA were performed using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, USA) and the GeneChip TM Hybridization, Wash and Stain Kit (Affymetrix) following the manufacturer's protocol. The puri ed and fragmented labeled cRNAs were hybridized onto the Affymetrix PrimeView TM Human Gene Expression microarray. After washing and staining, the arrays were scanned and analyzed using Affymetrix Scanner 3000 (Affymetrix). GeneChip Command Console Software (version 4.0, Affymetrix) and Genespring software (version 14.9, Agilent Technologies) was used to analyze the transcriptome data. Differentially expressed genes (DEGs) from The Cancer Genome Atlas (TCGA) were analyzed using DESeq2 package within R language (version 3.4.3).
9
Transcriptome analysis of Adv-hFGF23 in RC cells
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Total RNA (1 μg; evaluated by RIN value) was extracted from Adv-hFGF23-or Adv-βgal-transfected RC cells (n = 3 replicates for each) and used for generation of second-strand cDNA, and cRNA was amplified with the Oligo dT primer, biotinylated and fragmented with One-Cycle Target Labeling and control reagents (Affymetrix), followed by hybridization to the GeneChip Rat Genome 230 2.0 Array overnight according to the manufacturer's protocol.
10
Rat Liver and Lung Transcriptome Analysis
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Total RNA was isolated from liver and lung samples obtained independently from 5 rats in each anesthetic group using Trizol LS (Life Technologies Japan, Tokyo, Japan) and an RNeasy mini kit (Qiagen, Valencia, CA, USA) following the manufacturers' instructions. Gene expression was assessed using One-Cycle Target Labeling and Control Reagents and GeneChip Rat Genome 230 2.0 arrays (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions. The resulting digital image files were preprocessed using MAS5.0, and the results for each sample were normalized by dividing the value by the median value for all genes, excluding the control probe sets defined by Affymetrix that begin with the designation "AFFX-". Fold change (FC) and p-values for one-way ANNOVA were calculated using R scripts.
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