At the time of analysis, the right upper lobe from three to four mice per group was excised from OCT, placed in RNAlater (Fisher Scientific) for 5 min, and homogenized in 2 ml lysis buffer (QIAGEN, Valencia, CA) with zirconia-silica beads using a BeadBeater (BioSpec Products, Bartlesville, OK). RNA isolation was performed using the QIAGEN RNeasy kit according to the manufacturer's protocol with slight modifications (Barry et al., 2010 (link)). For gene expression analysis, samples were not pooled. Total RNA was assessed for quality with Agilent 2100 Bioanalyzer G2939A (Agilent Technologies, Santa Clara, CA) and Nanodrop 8000 spectrophotometer (Thermo Scientific–Nanodrop, Wilmington, DE). Hybridization targets were prepared with MessageAmp Premier RNA Amplification Kit (Applied Biosystems–Ambion, Austin, TX) from total RNA, hybridized to GeneChip Mouse Genome 430 2.0 arrays in Affymetrix GeneChip hybridization oven 645, washed in Affymetrix GeneChip Fluidics Station 450, and scanned with Affymetrix GeneChip Scanner 7G according to standard Affymetrix GeneChip Hybridization, Wash, and Stain protocols (Affymetrix, Santa Clara, CA).
Agilent 2100 bioanalyzer g2939a
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 2100 Bioanalyzer G2939A is a laboratory instrument designed for the analysis of biological samples. It utilizes microfluidic technology to perform automated electrophoresis and provide quantitative and qualitative assessment of DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Genechip fluidics station 450, by Thermo Fisher Scientific (3 mentions)
Affymetrix genechip scanner 7g, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by Qiagen (1 mentions)
Bead beater, by Biospec (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Agilent b scanner, by Agilent Technologies (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nugen ovation rna amplification system v2, by Tecan (1 mentions)
Affymetrix human genome u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
5 protocols using agilent 2100 bioanalyzer g2939a
1
RNA Extraction and Microarray Analysis in Mouse Lung
2
C. elegans Transcriptomic Profiling
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Total RNA was assessed for quality with an Agilent 2100 Bioanalyzer G2939A (Agilent Technologies, Santa Clara, CA) and a Nanodrop 8000 spectrophotomer (Thermo, Wilmington, DE). 100 ng of total RNA was converted to 1.65 µg Cy-3-labeled, linearly amplified cRNA using the Low Input Quick Amp (LIQA) Labeling One-Color Microarray-Based Gene Expression Analysis Kit (Agilent Technologies, Santa Clara, CA). cRNA was fragmented and added to 44 K feature Agilent C. elegans Gene Expression Microarray V2 slides (Agilent Technologies, Santa Clara, CA). Hybridization was performed in the Agilent rotisserie Hybridization Oven for 17 hours. Arrays were subsequently washed and scanned with the Agilent B scanner according to standard Agilent protocols (Agilent Technologies, Santa Clara, CA). Scanned data was log2 transformed and quantile normalized using Partek Genomics Suite (St. Louis, MO). Analysis of variance (ANOVA) t tests and fold-change calculations were also performed using Partek Genomics Suite (St. Louis, MO). For each of the 5 time points, 2 biological replicates were assessed. The microarray data was deposited in the Gene Expression Omnibus database: GSE54212.
3
Transcriptional Profiling of A2EN Cells Infected with tepP Mutant
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Approximately 0.8×106 A2EN cells were seeded per well in 6-well plates the day before experiment. Duplicate cell samples were mock infected, or infected with the tepP mutant strain transformed with empty vector or the vector harboring wild type tepP at an MOI of 10. Infections were synchronized by centrifugation at 3000 rpm for 30 min at 10°C, followed by an immediate shift to 37°C with pre-warmed cell culture media. Samples were collected at 4 hpi using QIAGEN RNeasy Plus Mini Kit (QIAGEN, Valencia, CA, USA) as described by the manufacturer. RNA integrity was assessed with Agilent 2100 Bioanalyzer G2939A (Agilent Technologies, Santa Clara, CA, USA) and quantified with a Nanodrop 8000 spectrophotometer (Thermo Scientific/Nanodrop, Wilmington, DE, USA). Hybridization targets were prepared with MessageAmp Premier RNA Amplification Kit (Applied Biosystems/Ambion, Austin, TX, USA) from total RNA, hybridized to GeneChip Human Genome U133A 2.0 arrays in Affymetrix GeneChip hybridization oven 645, washed in Affymetrix GeneChip Fluidics Station 450 and scanned with Affymetrix GeneChip Scanner 7G according to standard Affymetrix GeneChip Hybridization, Wash, and Stain protocols (Affymetrix, Santa Clara, CA, USA). Data analysis was performed using Partek Genomic Suite 6.6 (Partek Inc., Saint Louis, MO, USA).
4
Transcriptome Profiling via Affymetrix Microarrays
5
RNA Isolation, Microarray Processing, and Differential Gene Expression Analysis
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RNA was isolated using the QIAGEN RNeasy micro kit (QIAGEN). RNA was then sent to the UCLA Clinical Microarray Core (CMC) for processing. Total RNA was assessed for quality with Agilent 2100 Bioanalyzer G2939A (Agilent Technologies) and Nanodrop 8000 spectrophotometer (Thermo Scientific/Nanodrop). The samples were amplified using the NuGEN Ovation RNA Amplification System V2 (catalog no. 3100-A01; NuGEN Technologies Inc.) and were then hybridized to Affymetrix Human Genome U133 Plus 2.0 Array. Affymetrix GeneChip CEL files for the samples assayed were imported into the Affymetrix Expression Console software to perform gene level normalization and signal summarizations. Affymetrix Transcriptome Analysis Console (TAC) software was used to analyze the differential gene expression between the cell types. Robust multiarray average (RMA) values generated in the Affymetrix Expression Console software from this gene list were imported into Partek microarray data analysis software (Partek Inc.) and unsupervised hierarchical cluster analysis was performed on the various gene lists. For all results of differential gene expression analysis, statistical filters were applied: P < 0.05, FDR < 0.05, and |FC|>2. Within the differentially expressed gene set, QIAGEN's IPA (QIAGEN, www.qiagen.com/ingenuity ) was applied to identify enriched canonical pathways, diseases, and biological functions.
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