Affymetrix GeneChip Rat Genome U34A [RG_U34A]

Genetically determined susceptibility to neurodegeneration is associated with expression of inflammatory genes

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Gene expression profiling in rat lumbar spinal cord following ventral root avulsion in the two inbred rat strains.

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Transcription profiling of rat lumbar spinal cord following ventral root avulsion in two inbred strains to investigate susceptibility to neurodegeneration

Gene expression profiling in rat lumbar spinal cord following ventral root avulsion in the two inbred rat strains.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v3

Title: Affymetrix EukGE-WS2v3 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

Four samples of 2 microgram of total RNA from the same time point and strain were pooled and hybridized on the same microarray.

Total RNA from the ipsilateral L4 spinal cord segment (7-16 mg) of naïve and operated rats was extracted using TRIzol (GIBCO/BRL Life technologies, Rockville, MD) according to the supplier's instructions with the inclusion of Pellet paint (Novagen, Madison, WI) as co-precipitant. The RNA was purified with RNeasy Mini kit (Qiagen, Hilden, Germany) and concentrations determined spectrophotometrically. The RNA was precipitated by addition of ethanol and subsequently resuspended in RNase free water (0.7 microg/microl). The quality of the RNA was analysed by non-denaturing 1.1% agarose gel electrophoresis of 0.14 microg RNA from each pool. The RNA was visualised with SYBR Gold (Molecular Probes, Eugene, OR).

Labeling was performed according to the protocol in the Expression Analysis Technical Manual (701021 rev 1, Affymetrix, Santa Clara, CA).

Naïve animals used as controls were sacrificed with CO2 and perfused with cold PBS. Spinal cords were examined in a dissection microscope. The meninges were removed and the left L4 ventral quadrant dissected by dividing the cord vertically and horizontally at the level of the central canal. The tissue was subsequently snap frozen and stored at -80 C until further use.

Five days following unilateral ventral root avulsion the rats were sacrificed with CO2 and perfused with cold PBS. Spinal cords were examined in a dissection microscope to verify the completeness of the lesion and to exclude signs of hemorrhage, necrotic zones or direct damage to the spinal cord. The meninges were removed and the ipsilateral L4 ventral quadrant dissected by dividing the cord vertically and horizontally at the level of the central canal. The tissue was subsequently snap-frozen and stored at -80 C until further use.

Fourteen days following unilateral ventral root avulsion the rats were sacrificed with CO2 and perfused with cold PBS. Spinal cords were examined in a dissection microscope to verify the completeness of the lesion and to exclude signs of hemorrhage, necrotic zones or direct damage to the spinal cord. The meninges were removed and the ipsilateral L4 ventral quadrant dissected by dividing the cord vertically and horizontally at the level of the central canal. The tissue was subsequently snap-frozen and stored at -80 C until further use.

Transcription profiling of rat lumbar spinal cord following ventral root avulsion in two inbred strains to investigate susceptibility to neurodegeneration

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