For microarray analysis, HeLa cells were treated with VLX600 (6.5 μM) or FCCP (10 μM) for 12 h. Cells were then lysed and total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufacturer instructions. 500 nM total RNA was biotin labeled using the Flashtag™ Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA). Successful biotin labeling was confirmed using the included ELISA QC assay. Biotin labeled RNA was hybridized to GeneChip® miRNA 4.0 chips for 16 h in a GeneChip® Hybridization Oven 645, washed and stained using the GeneChip® Fluidics Station 450 using the FS450_0003 protocol and scanned with a GeneChip® Scanner 3000 7G (Affymetrix, Santa Clara, CA). Expression data was normalized using RMA-DABG analysis in the Affymetrix® Expression Console Software build 1.4.1.46. Changes in expression were visualized using Affymetrix® Transcriptome Analysis Console (TAC) Software version 3.1.0.5 (both Affymetrix, Santa Clara, CA).
Hybridization oven 645
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Hybridization Oven 645 is a laboratory equipment designed for controlled incubation of samples during hybridization processes. It maintains a stable temperature and environment for optimal hybridization conditions.
Automatically generated - may contain errors
Lab products found in correlation
Fluidics station 450, by Thermo Fisher Scientific (33 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (20 mentions)
Command console software 3, by Thermo Fisher Scientific (18 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (13 mentions)
Gene spring software 11, by Agilent Technologies (12 mentions)
Genechip 3 ivt express kit, by Thermo Fisher Scientific (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Rnase free dnase set, by Qiagen (7 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (6 mentions)
Rneasy micro kit, by Qiagen (5 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Command console software 4, by Thermo Fisher Scientific (4 mentions)
Genechip eukaryotic hybridization control kit, by Thermo Fisher Scientific (4 mentions)
Flashtag biotin hsr rna labeling kit, by Thermo Fisher Scientific (3 mentions)
Genechip 3 ivt plus reagent kit, by Thermo Fisher Scientific (3 mentions)
Expression console, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Affymetrix wt plus reagent kit, by Thermo Fisher Scientific (2 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (2 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (2 mentions)
42 protocols using hybridization oven 645
1
Microarray Analysis of HeLa Cells Treated with VLX600 or FCCP
2
Transcriptomic Analysis of ALPs and BLPs
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Gene expression profiles were obtained with the GeneChip Mouse Gene 2.0 ST Arrays with approximately 20–30 ng of total RNA obtained from ALPs and BLPs. Total RNA was amplified, labeled, and purified using the Encore biotin module of the NuGEN Ovation Pico WTA System V2 following the manufacturer's instructions to obtain biotin-labeled cDNA. Array hybridization and washes were performed using the GeneChip Hybridization, Wash and Stain Kit (Affymetrix) in a Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix). Slides were scanned by a GeneChip Scanner 3000 (Affymetrix) using the Command Console Software 3.1 (Affymetrix) with default settings. CEL files from the Mouse Gene 2.0 chips were normalized with the RMA algorithm in the Expression Console software.
3
Microarray Data Analysis Pipeline
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Total RNA was extracted and purified using RecoverAllTM Total Nucleic Acid Isolation (Cat#AM1975, Ambion, Austin, TX, US) following the manufacturer’s instructions. Then, the total RNA was amplified, labeled and purified by Affymetrix WT PLUS Reagent Kit (Cat#902280, Affymetrix, Santa Clara, CA, US), Ovation FFPE WTA System (Cat#3403, NuGEN, San Carlos, CA, US) and FL-Ovation™ cDNA Biotin Module V2 (Cat#4200, NuGEN, San Carlos, CA, US). Array hybridization and washing was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in a Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and a Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US). Arrays were scanned by Affymetrix GeneChip® Scanner 3000 (Cat#00-00213, Affymetrix, Santa Clara, CA, US) to obtain raw data. The raw data were normalized by Expression Console software of Affymetrix company. After normalization, the fold changes and p-values of different genes were calculated by the formula: foldchange = average (power (2, signal (FTC)))/average (power (2, signal (BTL))) and t-test separately. Differentially expressed genes (DEGs) were reported if the fold change was >2 or <0.5 and the p-value was smaller than 0.05. At last, Unsupervised consensus clustering was performed by R package “pheatmap” to obtain the heatmap of DEGs.
4
Transcriptomic Analysis of Space Plants
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Total RNA was extracted from the rosette leaves of space plants and their ground controls in PCB, then purified using the miRNeasy Mini Kit (Cat#217004, QIAGEN, GmBH, Dusseldorf, Germany) following the manufacturer’s instructions, and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, USA). RNA was amplified, labeled, and purified using the GeneChip 3′ IVT PLUS Reagent Kit (Cat#902416, Affymetrix, Santa Clara, CA, USA) following the manufacturer’s instructions to obtain biotin-labeled cRNA. The array hybridization and wash were performed using the GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, USA) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, USA) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, USA) following the manufacturer’s instructions.
5
Microarray Gene Expression Analysis Protocol
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In order to evaluate the integrity of the RNA, a microfluidic analysis was performed using an Agilent 2100 Bioanalyzer with the RNA6000 nano kit (Agilent Technologies, Palo Alto, CA, USA). For the microarray analysis, we used only RNA samples whose RNA integrity number (RIN) was greater than 8.5. The gene expression was analyzed using a GeneChip® Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) containing 764,885 probes (and supporting 28,869 genes). Target cDNA was prepared from 200 ng of total RNA with the Ambion® WT Expression kit (Ambion, Austin, TX, USA) and the Affymetrix® GeneChip® WT Terminal Labeling kit (Affymetrix). Hybridization to the microarrays, washing, staining and scanning were performed using the GeneChip® system (Affymetrix) composed of a Scanner 3000 7G Workstation Fluidics 450 and a Hybridization Oven 645. The scanned image data were processed using the Affymetrix® Expression Console™ Version 1.1. The fold-change for each gene was evaluated using a Gene Expression Analysis with the Partek® Genomics Suite 6.5 software program (Partech, Münster, Germany). Genes with an expression level greater than 2-fold or less than 0.5 were recognized as being significantly different.
6
Affymetrix Gene Expression Profiling
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Total RNA was extracted using TRIzol reagent (cat. no. 15596-018; Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions, and RNA integrity was checked against an RIN threshold in an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Qualified total RNA was further purified with an RNeasy Micro kit (cat. no. 74004; Qiagen, GmBH, Germany) and RNase-Free DNase Set (cat. no. 79254; Qiagen). Total RNA was then amplified, labeled and purified using a GeneChip 3′IVT Express Kit (cat. no. 901229; Affymetrix, Santa Clara, CA, USA) following the manufacturer's instructions to obtain biotin-labeled cRNA. For array hybridization, the hybridization procedure was performed using a GeneChip® Hybridization, Wash and Stain Kit (cat. no. 900720; Affymetrix) in a Hybridization Oven 645 (cat. no. 00-0331-220V) and Fluidics Station 450 (cat. no. 00-0079; Affymetrix) following the manufacturer's instructions. For data acquisition, slides were scanned with a GeneChip® Scanner 3000 (cat. no. 00-00212; Affymetrix) using Command Console Software 3.1 (Affymetrix) at default settings. Raw data were normalized by the MAS 5.0 algorithm using Gene Spring Software 11.0 (Agilent Technologies).
7
Microarray Analysis of ERT2-cre;Rhau Mice
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For microarray analysis, hearts were collected at 2 months after tamoxifen treatment of adult ERT2-cre;RhauF/F mice and controls (RhauF/F). Total RNA was extracted using TRIZOL Reagent as described previously, and RNA was assessed with an Agilent Bioanalyzer 2100 (Agilent technologies) to measure RNA integrity. Qualified total RNA was further purified with an RNeasy Micro Kit (QIAGEN, #74004) and an RNase-Free DNase Set (QIAGEN, #79254). Afterward, total RNA was amplified, labeled, and purified by using a GeneChip 3′IVT Express Kit (Affymetrix, #901229) following the manufacturer's instructions to obtain biotin-labeled cRNA. Array hybridization and washing were performed using a GeneChip Hybridization, Wash and Stain Kit (Affymetrix, #900720) in Hybridization Oven 645 (Affymetrix) and a Fluidics Station 450 (Affymetrix) according to the manufacturer's instructions. Slides were scanned by a GeneChip Scanner 3000 (Affymetrix) and Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized by the MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent Technologies). The data were analyzed by use of the eBioService System (Shanghai Biotechnology, China).
8
Poplar Genome Microarray Expression Analysis
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Array hybridization and washes were performed using the GeneChip Hybridization, Wash and Stain Kit (cat. #900720; Affymetrix, Santa Clara, CA) in a Hybridization Oven 645 (cat. #00-0331-220 V; Affymetrix) and Fluidics Station 450 (cat. #00-0079; Affymetrix) following the manufacturer’s instructions. The array contains 61,251 poplar probe sets (including seven rRNA probe sets), 12 poplar control probe sets, and 62 reporter probe sets. Slides were scanned by GeneChip Scanner 3000 (cat. #00-00212; Affymetrix) and Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized via the MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent Technologies). Due to the high technical consistency and reliability of microarrays, technical replications of hybridizations were not performed. cRNA labeling and hybridization to the Poplar Genome Array were performed by the ShanghaiBio Corporation in China. The expression data of all samples were uploaded to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ ) with accession number GSE47105.
9
Affymetrix Gene Expression Profiling
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Total RNA were amplified, labeled and purified using Affymetrix WT Amplication Kit (Affymetrix) and GeneChip WT Terminal Labeling Kit (Affymetrix) to obtain biotin-labeled cDNA. Array hybridization and wash was performed using GeneChip Hybridization Wash and Stain Kit (Affymetrix) in Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix). Slides were scanned by GeneChip Scanner 3000 (Affymetrix) and Command Console Software 3.1 (Affymetrix) with default settings. Raw data were normalized by Gene Spring Software 11.0 (Agilent Technologies, Palo Alto, CA, USA).
10
Whole Genome Expression Profiling by Affymetrix Microarray
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RNA extraction and purification, RNA amplification and labeling, array hybridization, and data acquisition were performed as described in the Affymetrix manufacturer's instructions. Affymetrix Human U133 Plus 2.0 whole genome chips were used (Santa Clara, CA, US). Array hybridization and wash was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer's instructions. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
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