Rats were subjected to experimental severe fluid percussion brain injury, with and without drug treatment, sham injury or were sacrificed with no treatment (naïve) and brains were removed 24 hours later, hippocampi microdissected and stored in RNA later. JM6 and PMI were administered by oral gavage and E33 administered subcutaneously.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted and purified using Ribopure (Ambion) RNA isolation
Label
Alexa-555
Label protocol
Labeled cRNA was prepared from total RNA samples. Briefly, the Poly(A)+ RNA population within total RNA was amplified using Arcturus RiboAmp HS reagents (Molecular Devices, Sunnyvale, CA). Alternatively, MessageAMP II (Applied Biosystems, Foster City, CA) was used. After a second round of reverse transcription, second-strand cDNA synthesis, and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase in the presence of Biotin-11-UTP. The quantity and quality of the cRNA was assayed by spectrophotometry and on the Agilent Bioanalyzer.
Hybridization protocol
1 µg of purified cRNA was fragmented to uniform size and applied to Agilent Whole Rat Genome microarrays (Agilent Technologies, Santa Clara, CA) in hybridization buffer. Arrays were hybridized at 65° C for 17 hrs. in a rotating incubator, washed at 37° C for 1 min, and stained with Streptavidin-Alexa555.
Scan protocol
Rinsed and dried arrays were scanned with an Agilent G2565 Microarray Scanner (Agilent Technologies, Santa Clara, CA) at 5 µm resolution.
Description
Gene expression of whole hippocampus 24hr post-injury
Data processing
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA).
