Moderate Fluid percussion injury (TBI), sham and Naive at various time points (24hr, 2 week,3,6 and 12 month)
Growth protocol
Rats were prepared for fluid percussion traumatic brain injury or sham injury (Naive rats had no anesthesia and were not handled in any way and gene expression in their brains serve as baseline data) and sacrificed 24 hr, 2 week, 3 month, 6 month and 1 year post-injury, hippocampi and cortex were obtained, and stored in RNA later.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted and purified using Ribopure (Ambion) RNA isolation. Quality and quantity of the Total RNA sample was assessed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies; Santa Clara, CA).
Label
Alexa-555
Label protocol
Labeled cRNA was prepared by linear amplification of the Poly(A)+ RNA population within the Total RNA sample. Briefly, Total RNA was reverse transcribed after priming with a DNA oligonucleotide containing the T7 RNA polymerase promoter 5’ to a d(T)24 sequence. After second-strand cDNA synthesis and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase. 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.
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).
