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| Status |
Public on Nov 10, 2010 |
| Title |
tTA/TDP-DeltaNLS_3275_14202_2_MOE430A_v2.0 |
| Sample type |
RNA |
| |
|
| Source name |
Cerebral cortex
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: mixed C57BL/6J x C3HeJ
mouse line: NLS4
genotype: +/+
Sex: M
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen tissue samples were homogenized for 30-60 seconds in TRIzol (Invitrogen) using an Omni Homogenizer with disposable probes (Omni International). RNA was then isolated from homogenates using TRIzol phase separation as per manufacturer protocol. Total RNA quality and mass were determined using an Agilent Bioanalyzer 2100 and a Nanodrop spectrophotometer. All samples showed distinct 28S and 18S ribosomal RNA bands with RINs of 7 or greater. A260/280 ratios ranged from 2.00 – 2.04, with RNA concentrations ranging from 123 – 221ng/uL.
|
| Label |
biotin
|
| Label protocol |
All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 3ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotin using the Affymetrix 1-Cycle Target kit.
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| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99oC for 5 min and hybridized for 16 h at 45oC to the mouse genome MOE430A.v2 array from Affymetrix. Hybridization cocktails included 10ug of cRNA sample and the spiked controls provided in the Affymetrix Eukaryotic Control Kit. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
Hybridization signals were collected using a GeneChip 3000 7G scanner.
|
| Description |
Genotype: Presence (+) or absence (-) of transgene, with the first value corresponding the TDP-43 transgene and the second value corresponding to the tTA transgene. For example, +/- indicates the presence of the TDP-43 transgene and the absence of the tTA transgene
Line: Mouse line according to nomenclature in Igaz et al., JCI, 2012
|
| Data processing |
The Partek Genomics Suite was used to perform microarray data analysis and to generate graphics (Partek GS, St. Louis, MO). Affymetrix array raw fluorescence intensity measures of gene expression were normalized and quantified using robust multi-array analysis. To identify genes differentially expressed between groups, we employed an ANOVA, followed by pairwise contrasts between the groups of interest. Our model corrected for gender. To correct for multiple hypothesis testing, we calculated false discovery rates (FDR) using the Benjamini-Hochberg method. Principal components analyses and hierarchical cluster analyses (Pearson correlation coefficient) were employed to evaluate patterns of global gene expression. Differentially expressed genes were additionally subjected to biological pathway analysis using the Gene Ontology database to find pathways enriched in dysregulated genes; enrichment scores are the negative log10 chi-square p-value.
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| |
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| Submission date |
Nov 08, 2010 |
| Last update date |
Nov 10, 2010 |
| Contact name |
Virginia M.-Y. Lee |
| Organization name |
University of Pennsylvania
|
| Street address |
3rd Floor Maloney Building; 3600 Spruce St
|
| City |
Philadelphia |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
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