|
| Status |
Public on May 11, 2009 |
| Title |
anx/anx versus wild type hypothalamus replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
hypothalamus
|
| Organism |
Mus musculus |
| Characteristics |
Hypothalamus tissue from individual postnatal day 16 weanling mice anx/anx mouse (B6C3Fe a/a-anx/J strain)
|
| Growth protocol |
Homozygous anorexia mice were produced from heterozygous breeder pairs (B6C3Fe a/a-anx/J) obtained from the Jackson Laboratory. Postnatal day 16 weanling mice housed with their parents were separated into two groups: anx/anx and control mice. anx/anx mice were identified by their reduced body weight, body tremor, and mild hyperactivity. Because homozygous and heterozygous mice cannot be phenotypically distinguished from wild types, control groups probably contained both genotypes (wt/wt or wt/anx). anx/anx and control mice were allowed ad libitum access to the mother, food, and water. Three anx/anx and three control mice animals were anesthetized with isofluorane, decapitated and hypothalamus was dissected. Tissues were immediately frozen with liquid nitrogen and stored until the experiments were performed. Sex was determined by a simplex PCR assay as described (Clapcote and Roder 2005).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the tissue using Trizol (Invitrogen), following manufracturer’s recommendations. RNA quantification was performed through a Nanodrop spectrophotometer, and their quality was assessed using the Bioanalyzer 2100 nanoelectrophoresis Lab-on-a–chip system (Agilent, Wilmington, DE). All RNAs used had RIN (RNA integrity number) ranging between 8.4 and 8.9, and 28S/18S ratios between 1.3 and 1.6.
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).
|
| |
|
| Channel 2 |
| Source name |
hypothalamus
|
| Organism |
Mus musculus |
| Characteristics |
Hypothalamus tissue from individual postnatal day 16 weanling mice wild type mouse (B6C3Fe a/a-anx/J strain)
|
| Growth protocol |
Homozygous anorexia mice were produced from heterozygous breeder pairs (B6C3Fe a/a-anx/J) obtained from the Jackson Laboratory. Postnatal day 16 weanling mice housed with their parents were separated into two groups: anx/anx and control mice. anx/anx mice were identified by their reduced body weight, body tremor, and mild hyperactivity. Because homozygous and heterozygous mice cannot be phenotypically distinguished from wild types, control groups probably contained both genotypes (wt/wt or wt/anx). anx/anx and control mice were allowed ad libitum access to the mother, food, and water. Three anx/anx and three control mice animals were anesthetized with isofluorane, decapitated and hypothalamus was dissected. Tissues were immediately frozen with liquid nitrogen and stored until the experiments were performed. Sex was determined by a simplex PCR assay as described (Clapcote and Roder 2005).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the tissue using Trizol (Invitrogen), following manufracturer’s recommendations. RNA quantification was performed through a Nanodrop spectrophotometer, and their quality was assessed using the Bioanalyzer 2100 nanoelectrophoresis Lab-on-a–chip system (Agilent, Wilmington, DE). All RNAs used had RIN (RNA integrity number) ranging between 8.4 and 8.9, and 28S/18S ratios between 1.3 and 1.6.
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).
|
| |
|
| |
| Hybridization protocol |
Three biological replicate experiments were performed, each comparing anx/anx verus wild type tissue. For two of the three biological replicates each experimental pair of labelled samples was co-hybridized on two separate microarrays with dye swapping to correct for dye bias effects, whereas the third was only hybridized once. Thus, five microarray hybridizations were processed.
|
| Scan protocol |
Fluorescent images were obtained using an Agilent G2565BA scanner at 100% PMT 100% laser power settings and quantified through the GenePix 6.0 software (Axon, Molecular Devices, Sunnywale, CA) using the irregular feature finding option.
|
| Description |
Hypothalamus biological replicate 2 of 3. Technical replicate 1 of 2. anx/anx versus wild type littermate comparison.
|
| Data processing |
Extracted raw data were filtered and normalized using the Limma package developed within the Bioconductor project in the R statistical programming environment. The two channels were balanced by lowess normalization using 0.3 as the span parameter with reduced weights in control and poor quality spots, followed by scaling across chips. Target genes were considered as differentially expressed when above 0.001 false discovery rate (FDR), using significant analysis of microarrays (SAM) test implemented in the samr library in R (Tusher et al 2001). All quantitative and statistical analyses were performed using using Limma package in the R. environment (Smyth 2004).
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| |
|
| Submission date |
May 13, 2008 |
| Last update date |
May 13, 2008 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE11426 |
Hypothalamus transcriptome profile suggests an anorexia-cachexia syndrome in anx/anx mouse model |
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