Following sacrifice, mice were perfused with 20mL PBS via the heart in order to remove blood and other non-adherent cells from the brain microvasculature. Brains were isolated, halved and preserved in either RNAlater (Qiagen, Doncaster, Australia).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from brain tissue using TRIzol reagent (Invitrogen, Mount Waverley, Australia), according to manufacturer’s instructions, and contaminants were removed by passing the RNA over RNeasy mini-columns with on-column DNase treatment (Qiagen). RNA 6000 Nano Assay Kits (Agilent Technologies, Forest Hill, Australia) were used to assess RNA quality and integrity on a Bioanalyzer 2100 (Agilent Technologies). Samples were included in the study based on 260:280nm absorbance readings. Equal quantities of six samples were pooled for microarray analysis.
Label
R-Phycoerythrin
Label protocol
Pooled RNA samples were converted to cDNA and antisense cRNA, labelled and hybridized to GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix, Surrey Hills, Australia) by the Australian Genome Research Facility (Parkville, Australia), according to Affymetrix protocols.
Description
Arrays were scanned using the GeneChip Scanner 3000 (Affymetrix) and GeneChip Operating Software v1.1.1 (Affymetrix). Normalisation and initial analyses were carried out in GeneSpring v7 (Agilent Technologies). Values below 0.01 were set to 0.01. Each measurement was divided by the 50th percentile of all measurements in that sample. The data was filtered for genes flagged as present, which had at least an expression level of 50. Following this, a threshold of 2.5 fold up-regulation or down-regulation of genes was set.
