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Status |
Public on Nov 01, 2009 |
Title |
Olfactory_Epithelium_Rat-32 |
Sample type |
RNA |
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Source name |
Olfactory epithelium of rat 32 (litter4)
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Organism |
Rattus norvegicus |
Characteristics |
strain: Brown Norway tissue: Olfactory epithelium gender: Not known litter: Litter4 age: 3-5 days
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using the Nucleospin RNA kit according to the manufacturer’s (Macherey-Nagel) instructions, which included an in-column DNase treatment before RNA elution to ensure the absence of genomic DNA. Recovered RNA was quantified using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies), and RNA integrity was assessed with RNA 6000 Nano LabChip kit using the Agilent 2100 Bioanalyzer (Agilent Technologies). Only RNA samples with an RNA Integrity Number (RIN) greater than 8.5 were used for further analysis (RNA profiling analysis and semi quantitative reverse transcription PCR analysis). Because of the application of this strict quality threshold, the left sample from one adult olfactory epithelium was not studied.
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Label |
Cy3
|
Label protocol |
RNA samples were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification kit (p/n 5184-3523) according to the manufacturer’s instructions. Briefly, 350 ng of total RNA was used as template and copied into cDNA by reverse transcription and concomitant amplification with T7-polymerase; cyanine-3 (Cy3)-labeled CTP was used for labeling. Cy3 labeling was monitored with a Nanodrop ND-1000 spectrophotometer and was found to be between 1.2 and 1.9 pmol/μl.
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Hybridization protocol |
Hybridization was performed using the Agilent Gene Expression Hybridization kit (p/n 5188-5242), following the manufacturer’s instructions. Briefly, 1650 ng of labeled cRNA was mixed with Hybridization Buffer and Blocking Agent and subjected to fragmentation (by incubation for 30 min at 60°C in the dark). Hybridizations onto 4x44K Whole Rat Genome 60-mer oligonucleotide microarrays (G4131F) (Agilent Technologies) were performed in a rotary oven (65°C, 17 h and 10 rpm) in the dark. Slides were disassembled and washed in Gene Expression wash Buffers I and II according to the manufacturer’s instructions, and dried using a nitrogen-filled air gun before scanning.
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Scan protocol |
Microarrays were scanned with a dynamic autofocus microarray scanner (Agilent DNA Microarray Scanner), using Agilent parameters. Feature Extraction software version 9.5 was used to extract and analyze the signals.
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Description |
OE_rat32_litter4.txt
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Data processing |
Array results were analyzed using GeneSpring GX software version 7.3 (Agilent Technologies), via the Enhanced Agilent Feature Extraction Import Preprocessor. Data were normalized in two steps using the algorithms supplied with the Feature Extraction software. Data were first transformed to convert any negative value to 0.01, then normalization was performed by using a per-chip 50th percentile method that normalizes each chip to its median, allowing comparison between chips. A second normalization step was applied to the results for each gene across all the arrays in the study (normalize to median): the median of all the values obtained for a given gene was calculated and used as the normalization standard for that gene, such that, regardless of absolute differences in the expression of the various genes, they are placed on the same scale for comparison.
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Submission date |
May 05, 2009 |
Last update date |
May 05, 2009 |
Contact name |
Francis Galibert |
E-mail(s) |
francis.galibert@univ-rennes1.fr
|
Organization name |
CNRS UMR6061/Université Rennes1
|
Department |
Génétique et Developpement
|
Street address |
2 av du Pr Léon Bernard
|
City |
RENNES |
ZIP/Postal code |
35043 |
Country |
France |
|
|
Platform ID |
GPL4135 |
Series (2) |
GSE15953 |
RNA profiles of rat olfactory epithelia : individual and age related variations (age experiment) |
GSE15954 |
RNA profiles of rat olfactory epithelia : individual and age related variations |
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