|
| Status |
Public on Jun 01, 2011 |
| Title |
28_2_18H_AO |
| Sample type |
RNA |
| |
|
| Source name |
PBMC from blood, oleic acid, incubation 18H
|
| Organism |
Mus musculus |
| Characteristics |
genotype: wild-type
gender: female
strain: C57Bl/6J
age: 7 weeks old
tissue: PBMC
|
| Treatment protocol |
Femelle C57Bl/6J mice were anesthetized with 5% Xylazine – 20% Ketamine (0,1ml/10g). We injected 20 µl of oleic acid (1,2µl/g body weight, sigma #27728-1L-R) in the mouse. Administration of physiological serum and OA was done through the tail vein with a 0.3-ml insulin syringe (BD #320837).
|
| Growth protocol |
Mices were housed in a specific pathogen-free animal facility. All experimental procedures involving animals were approved by the veterinary office of the Ministry of Agriculture, France (authorization number: 13-27).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from lung was extracted using Mouse RiboPure-Blood RNA Isolation Kit (Ambion #AM1951) and amplified using MessageAmpII aRNA Amplification Kit (Ambion #AM1751). The quality of RNA was confirmed on a Agilent Bioanalyser 2100 using RNA NanoChips, and the concentration of RNA was dertermined by reading absorbance at 260/280 nm.
|
| Label |
α-dCTP 33P
|
| Label protocol |
cDNA were designed and prepared as described in J Immunol. 2004 Nov 15;173(10):6109-18. A general survey of thymocyte differentiation by transcriptional analysis of knockout mouse models.
Puthier D, Joly F, Irla M, Saade M, Victorero G, Loriod B, Nguyen C.
Labelling protocol is also published in « DNA microarrays: Gene expression applications » B. Jordan(ed.), Springer-Verlag Berlin Heidelberg Newyork, ISBN: 3-540-41508-4- chapter 4 – pp: 78-80.
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| |
|
| Hybridization protocol |
cDNA were designed and prepared as described in J Immunol. 2004 Nov 15;173(10):6109-18. A general survey of thymocyte differentiation by transcriptional analysis of knockout mouse models.
Puthier D, Joly F, Irla M, Saade M, Victorero G, Loriod B, Nguyen C.
Hybridization protocol is also published in « DNA microarrays: Gene expression applications » B. Jordan(ed.), Springer-Verlag Berlin Heidelberg Newyork, ISBN: 3-540-41508-4- chapter 4 – pp:80-82.
|
| Scan protocol |
Image acquisition was performed with a Fuji BAS 1500 system.
|
| Description |
The MessageAmp procedure is based on antisense RNA (aRNA) amplification first described by Van Gelder and Eberwine, and involves a series of enzymatic reactions resulting in linear amplification of exceedingly small amounts of RNA for use in array analysis. Unlike exponential RNA amplification methods, such as NASBA and RT-PCR, aRNA amplification maintains representation of the starting mRNA population. The procedure begins with total or poly(A) RNA that is reverse transcribed using a primer containing both oligo(dT) and a T7 RNA polymerase promoter sequence (see schematic at left). After first-strand synthesis, the reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during a second-strand synthesis reaction that produces a double-stranded cDNA template for transcription. Contaminating rRNA, mRNA fragments and primers are removed and the cDNA template is then used in a large scale in vitro transcription reaction to produce linearly amplified aRNA. For increased yields, the aRNA can also be used as template for cDNA synthesis followed by a second round of amplification using MessageAmp.
|
| Data processing |
Hybridation signals were quantified using the Bzscan2 software. All images were carefully inspected and spots with overestimated intensities due to neighborhood effects were manually excluded. We used the NylonArray library for R locally developped by A. Bergon and D. Puthier. This package contains functions to perform diagnosis and normalization of nylon microarray data. The sample datasets were corrected for neighborhood effects and local background as described by F. Lopez et al. Quantile normalization was applied to sample data to correct for global intensity and dispersion. At each time-point, gene expressions in control samples (physiological serum injection) were substracted from gene expression in OA samples.
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| |
|
| Submission date |
Nov 13, 2009 |
| Last update date |
Jun 01, 2011 |
| Contact name |
Isabelle Lesur Kupin |
| E-mail(s) |
lesur@tagc.univ-mrs.fr
|
| Phone |
(+33)4 91 82 87 11
|
| Fax |
(+33)4 91 82 87 01
|
| Organization name |
INSERM U928
|
| Department |
Bioinformatics
|
| Lab |
TAGC
|
| Street address |
163 av. de Luminy - case 928
|
| City |
Marseille |
| ZIP/Postal code |
13288 |
| Country |
France |
| |
|
| Platform ID |
GPL9478 |
| Series (1) |
| GSE19030 |
Gene Expression Profiles of PBMC Characterize Inflammation Stages in the Acute Lung Injury in Mice |
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