genotype/variation: wildtype age: 3 months tissue: pool of whole brain from 2 mice
Biomaterial provider
European Brain Research Institute (EBRI) “Rita Levi-Montalcini”,Genomics Facility, Roma, Italy
Growth protocol
AD11 transgenic mice (Ruberti et al., 2000) express a recombinant version of the monoclonal antibody mAb alfaD11 that specifically recognizes and neutralizes NGF (Cattaneo et al., 1988; Covaceuszach et al., 2008). AD11 mice were obtained by crossing two lines: one expressing only the Heavy VH chain and another expressing only the light VK chain of the antibody. The mouse line expressing only the VH chain (valled VH mice) were used as transgenic control mice, that are consistently negative with respect to all the neurodegeneration markers across lifespan.
Extracted molecule
total RNA
Extraction protocol
Hippocampus (HP), cortex (CTX) and basal forebrain (BFB) of the right hemisphere were dissected from the brains of freshly sacrificed mice AD11 and control VH, using RNAlater (Qiagen). A total of 60 female AD11 mice and 59 female control VH (5 per group) mice were used for this study: 5 AD11 and 4-5 control VH mice for each time point (1,3,6,15 months of age) and brain area(HP, CTX, BFB). Total RNA was isolated from these brain areas using Trizol (Invitrogen) and DNAse treated by Qiagen columns. Quality and integrity of each sample was checked using the Agilent BioAnalyzer 2100 (Agilent RNA 6000 nano kit): samples with a RNA Integrity Number (RIN) index lower than 8.0 were discarded. All the experimental steps involving the labelling, hybridization and washing of the samples were done following the Agilent protocol. Aliquots from the same RNA sample, prepared (and pooled) from 2 whole brains of wild type mice, were used in all hybridizations as a Reference sample on Channel 1 (Cy3 labelling) to reduce and control the experimental variability.
Label
Cy3
Label protocol
500ng of Poly A+RNA were labelled, using Cyanine 3-CTP or Cyanine 5-CTP (Perkin Elmer) in separate labelling reactions, with the Agilent Low Imput Linear Amplification Kit and purified with Qiagen’s RNeasy mini spin columns
European Brain Research Institute (EBRI) “Rita Levi-Montalcini”,Genomics Facility, Roma, Italy
Growth protocol
AD11 transgenic mice (Ruberti et al., 2000) express a recombinant version of the monoclonal antibody mAb alfaD11 that specifically recognizes and neutralizes NGF (Cattaneo et al., 1988; Covaceuszach et al., 2008). AD11 mice were obtained by crossing two lines: one expressing only the Heavy VH chain and another expressing only the light VK chain of the antibody. The mouse line expressing only the VH chain (valled VH mice) were used as transgenic control mice, that are consistently negative with respect to all the neurodegeneration markers across lifespan.
Extracted molecule
total RNA
Extraction protocol
Hippocampus (HP), cortex (CTX) and basal forebrain (BFB) of the right hemisphere were dissected from the brains of freshly sacrificed mice AD11 and control VH, using RNAlater (Qiagen). A total of 60 female AD11 mice and 59 female control VH (5 per group) mice were used for this study: 5 AD11 and 4-5 control VH mice for each time point (1,3,6,15 months of age) and brain area(HP, CTX, BFB). Total RNA was isolated from these brain areas using Trizol (Invitrogen) and DNAse treated by Qiagen columns. Quality and integrity of each sample was checked using the Agilent BioAnalyzer 2100 (Agilent RNA 6000 nano kit): samples with a RNA Integrity Number (RIN) index lower than 8.0 were discarded. All the experimental steps involving the labelling, hybridization and washing of the samples were done following the Agilent protocol. Aliquots from the same RNA sample, prepared (and pooled) from 2 whole brains of wild type mice, were used in all hybridizations as a Reference sample on Channel 1 (Cy3 labelling) to reduce and control the experimental variability.
Label
Cy5
Label protocol
500ng of Poly A+RNA were labelled, using Cyanine 3-CTP or Cyanine 5-CTP (Perkin Elmer) in separate labelling reactions, with the Agilent Low Imput Linear Amplification Kit and purified with Qiagen’s RNeasy mini spin columns
Hybridization protocol
Microarray hybridizations were carried out in Agilent’s SureHyb Hybridization Chambers containing 825 ng of Cyanine 3-labelled and 825 ng of Cyanine 5-labelled cRNA per hybridization. The hybridization reactions were performed at 65°C for 17 hours using Agilent’s Gene Expression Hybridization Kit with the 4X44K whole mouse genome chip (G4122F). After hybridization, slides were washed with Agilent Gene Expression Wash Buffer 1, Gene Expression Wash Buffer 2 and Acetonitrile for one minute at room temperature.
Scan protocol
Chips were scanned on an Agilent G2505B scanner. Images were quantified using Agilent Feature Extraction Software (version 9.1) and the GE2-v4_91 extraction protocol.
Data processing
Raw data filtering was performed in Microsoft Excel using any of the following criteria to discard spots: spots with more than 5% of saturated pixel in any of the two channels, spots flagged as “not found” by the Feature Extraction in any of the two channels, spots with a Signal/Noise ratio smaller than 3 in any of the two channels, where Signal = (median of the spot - median spot background level) and Noise is the IQR (interQuantileRange) of the median spot background. Data analysis was performed with R-Bionductor, Agilent GeneSpring GX ver 7.3 and Microsoft Excel. Data analysis was performed on filtered data using Agilent GeneSpring GX 7.3 and Microsoft Excel. Each array was normalized by the Lowess algorithm within GeneSpring, using 20% of data as smoothing window.
Gene expression profiling of the AD11 anti-NGF transgenic mouse model of neurodegeneration at 1, 3, 6, 15 months of age
Data table header descriptions
ID_REF
VALUE
log2 Lowess normalized ratio (Cy5/Cy3) for each single sample. Only filtered features were normalized. Since often different probes match the same gene symbol, ANOVA was used to select, for each gene, only the probe that allow the best discrimination between control and AD11 classes. A final filtered dataset with 16515 transcripts (probes) was obtained.
PRE_VALUE
Lowess normalized ratio (Cy5/Cy3) for each single sample. Only filtered features were normalized. Since often different probes match the same gene symbol, ANOVA was used to select, for each gene, only the probe that allow the best discrimination between control and AD11 classes. A final filtered dataset with 16515 transcripts (probes) was obtained.
