RNA samples were isolated from the hippocampus (HC) using TRI-Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The total RNA pellet was dissolved in nuclease-free water and RNA quality and quantity were assessed by Agilent Bioanalyzer 2100 analysis (Agilent, Santa Clara, CA). RNA with RNA Integrity Number (RIN) above 8 was used for microarray and sequencing. Gene expression was analyzed with GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA), which comprises over 45,000 probe sets representing approximately 28,700 well-characterized mouse genes. For each gene, eleven pairs of oligonucleotide probes were synthesized in situ on the arrays.
Agilent 2100 bioanalyzer analysis
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 2100 Bioanalyzer is a microfluidics-based platform that provides automated electrophoretic separation and detection of DNA, RNA, and proteins in a compact and integrated system. It enables detailed analysis of sample quality and quantity in a fast and user-friendly manner.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Messageamp 2 biotin enhanced arna kit, by Thermo Fisher Scientific (3 mentions)
Expression console software, by Thermo Fisher Scientific (3 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Poly a rna controls, by Thermo Fisher Scientific (2 mentions)
Genechip rat genome 230 2.0 array, by Thermo Fisher Scientific (2 mentions)
Genechip mouse genome 430 2.0 array, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Genechip microrna 4.0 array, by Thermo Fisher Scientific (1 mentions)
Expression console 1, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Spss statistics software version 24, by IBM (1 mentions)
Nanodrop analysis, by Thermo Fisher Scientific (1 mentions)
Qubit analysis, by Thermo Fisher Scientific (1 mentions)
12 protocols using agilent 2100 bioanalyzer analysis
1
Hippocampal RNA Isolation and Expression Analysis
2
Microarray Analysis of miRNA Expression
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Total RNA was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA USA) according to the manufacturer’s instructions. RNA quality and quantity was assessed by Agilent bioanalyzer 2100 analysis (Agilent Technologies, CA, USA).
Starting with 250 ng of total RNA, the labeling process begins with poly-A tailing of each RNA strand using poly-A polymerase, followed by ligation of biotin-labeled 3DNA dendrimer. Biotinylated RNA strands were hybridized at 48 °C for 18 h on the GeneChip microRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The GeneChip microRNA 4.0 Array was washed and stained in the Fluidics Station 450 (Affymetrix, CA, USA). Amplified fluorescence signals by the branched structure of 3DNA dendrimer were scanned using GeneChip Scanner 3000 7G (Affymetrix).
The arrays were analyzed using an Agilent scanner with associated software. The expression levels of miRNAs were calculated with Expression Console 1.4 (Affymetrix, Santa Clara, CA, USA). Relative signal intensities for each miRNA were generated using the Robust Multi-Array Average algorithm. Target prediction were analyzed using the miRBase, miRDB, TargetScan and microRNA.org DB.
Starting with 250 ng of total RNA, the labeling process begins with poly-A tailing of each RNA strand using poly-A polymerase, followed by ligation of biotin-labeled 3DNA dendrimer. Biotinylated RNA strands were hybridized at 48 °C for 18 h on the GeneChip microRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The GeneChip microRNA 4.0 Array was washed and stained in the Fluidics Station 450 (Affymetrix, CA, USA). Amplified fluorescence signals by the branched structure of 3DNA dendrimer were scanned using GeneChip Scanner 3000 7G (Affymetrix).
The arrays were analyzed using an Agilent scanner with associated software. The expression levels of miRNAs were calculated with Expression Console 1.4 (Affymetrix, Santa Clara, CA, USA). Relative signal intensities for each miRNA were generated using the Robust Multi-Array Average algorithm. Target prediction were analyzed using the miRBase, miRDB, TargetScan and microRNA.org DB.
3
Trigeminal Ganglia Gene Expression
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Two days following the placement of coil springs, the mice were anesthetized
using ketamine and xylazine and the TG in the ipsilateral side were harvested
from the RTX- or vehicle-injected mice. We chose day 2 since the maximum
pain-like behavior was observed on day 1 through day 3 in our previous study,3 (link) and we presumed that changes of gene expression in TG could occur during
days 1 to 3. We also assumed that gene expression changes on day 2 represented
changes occurring during day 1 to 3 and might contribute to pain-like behaviors
not only on day 2 but also on day 1 and 3. A total of sixteen dissected TG were
stored in RNAlater (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted
using Trizol (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy kit
(Qiagen, Germantown, MD, USA), which included a DNase treatment for removing
genomic DNA. RNA integrity was evaluated by Agilent 2100 Bioanalyzer analysis
(Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number of all
samples was greater than 8.5.
using ketamine and xylazine and the TG in the ipsilateral side were harvested
from the RTX- or vehicle-injected mice. We chose day 2 since the maximum
pain-like behavior was observed on day 1 through day 3 in our previous study,3 (link) and we presumed that changes of gene expression in TG could occur during
days 1 to 3. We also assumed that gene expression changes on day 2 represented
changes occurring during day 1 to 3 and might contribute to pain-like behaviors
not only on day 2 but also on day 1 and 3. A total of sixteen dissected TG were
stored in RNAlater (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted
using Trizol (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy kit
(Qiagen, Germantown, MD, USA), which included a DNase treatment for removing
genomic DNA. RNA integrity was evaluated by Agilent 2100 Bioanalyzer analysis
(Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number of all
samples was greater than 8.5.
4
Comprehensive RNA Profiling of B-cell Lymphomas
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Total RNA was extracted using a modified protocol combining TRIzol Reagent (Invitrogen, Paisley, UK) and mirVana miRNA Isolation Kit (Ambion/ThermoFisher Scientific, Grand Island, NY) as previously described [22 (link)]. RNA quality and concentration was determined by Agilent 2100 Bioanalyzer analysis (Agilent Technologies, Santa Clara, CA) and NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific), respectively. miRNA expression profiling was performed using GeneChip miRNA 1.0.2 arrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s protocol. The cell lines DB, FARAGE, OCI-Ly3, OCI-Ly7, OCI-Ly8, OCI-Ly19, NU-DHL-1, RIVA, and U2932 were prepared for hybridization using Flashtag HSR kit from Genesphere (Genesphere, Hatfield, PA) whereas HBL-1, MC-116, NU-DUL-1, SU-DHL-4, SU-DHL-5, and SU-DHL-8 were prepared using Fashtag Biotin HSR RNA labeling kit (Affymetrix) [14 (link)]. For GEP, RNA was labeled and hybridized to Affymetrix GeneChip Human Genome U133 (HG-U133) Plus 2.0 arrays, as described by the manufacturer. Generated miRNA and HG-U133 CEL files are deposited at NCBI GEO repository GSE72648 and GSE109027, respectively. The data comply with MIAME requirements [23 (link)].
5
Biotinylated antisense cRNA amplification
6
Illumina RNA-seq Library Preparation
7
Biotinylated antisense cRNA amplification
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Biotinylated antisense cRNA was prepared by single round in vitro amplification of 0.9 µg input RNA using the MessageAmp™ II-Biotin Enhanced aRNA kit (Ambion) according to the manufacturer’s instructions (the in vitro transcription reaction was performed at 37°C for 14 hours). Poly-A RNA controls (Affymetrix, Santa Clara, CA) were spiked into each reaction. Fragmented cRNA sample quality was confirmed using 2% agarose gel electrophoresis and Agilent 2100 Bioanalyzer analysis. Samples were hybridized to Affymetrix GeneChip® Rat Genome 230 2.0 arrays at 45°C for 16 hours. Post-processing was performing using the GeneChip® Fluidics Station 450, arrays were scanned using the GC3000 G7 scanner, and fluorescent intensity data were background-corrected and extracted using Expression Console™ software (Affymetrix). All hybridization, post-processing and scanning procedures were performed according to Affymetrix protocols; all control parameters were within the manufacturer’s guidelines. Microarray data have been deposited with the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ ) under accession number GSE62204.
8
Comprehensive RNA Extraction and RNA-seq Analysis
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Total RNA was extracted using a modified protocol combining TRIzol Reagent (Invitrogen, Paisley, UK) and mirVana miRNA Isolation Kit (Ambion/ThermoFisher Scientific, Grand Island, NY, USA), as previously described [105 (link)]. The RNA quality and concentration of each sample were determined by Agilent 2100 Bioanalyzer analysis (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific), respectively. The total RNA of each sample was sent to AROS Applied Biotechnology AS (Aarhus, Denmark) for poly-A selected, pair-end RNA-seq via Illumina HiSeq 2000 platform. The generated and analyzed data were deposited in the Gene Expression Omnibus (accession ID: GSE159852)
9
RNA Quality Assessment for Library Preparation
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Total RNA samples were subjected to three quality control analyses prior to library construction: (A) NanoDrop analysis (Thermo Fisher, United States) to assess RNA purity (OD260/OD280; OD260/OD230); (B) Qubit analysis (Thermo Fisher, United States) to determine the RNA yield; and (C) Agilent 2100 Bio-analyzer analysis (Agilent Technologies) to verify RNA integrity. The significance of the results was determined using analysis of variance (ANOVA) and SPSS Statistics software, version 24.0 (IBM, United States).
10
Affymetrix GeneChip Rat Genome 230 2.0 Analysis
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Biotinylated antisense cRNA was prepared by single-round in vitro amplification of 0.9 μg input RNA using the MessageAmp II-Biotin Enhanced aRNA kit (Ambion) according to the manufacturer’s instructions (the in vitro transcription reaction was performed at 37°C for 14 h). Polyadenylated RNA controls (Affymetrix) were spiked into each reaction. Fragmented cRNA sample quality was confirmed using 2% agarose gel electrophoresis and Agilent 2100 Bioanalyzer analysis. Samples were hybridized to Affymetrix GeneChip Rat Genome 230 2.0 arrays at 45°C for 16 h. Post-processing was performing using the GeneChip Fluidics Station 450, arrays were scanned using the GC3000 G7 scanner, and fluorescent intensity data were background-corrected and extracted using Expression Console software (Affymetrix). All hybridization, post-processing, and scanning procedures were performed according to Affymetrix protocols; all control parameters were within the manufacturer’s guidelines. Microarray data are available from the Gene Expression Omnibus (GEO): GSE62204 (https://www.ncbi.nlm.nih.gov/geo/ ).
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