Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.
BACKGROUND
Rat oligonucleotide microarrays were used to detect changes in gene expression in the dorsal root ganglion (DRG) 3 days following sciatic nerve transection (axotomy). Two comparisons were made using two sets of triplicate microarrays, naïve versus naïve and naïve versus axotomy.


RESULTS
Microarray variability was assessed using the naïve versus naïve comparison. These results support use of a P < 0.05 significance threshold for detecting regulated genes, despite the large number of hypothesis tests required. For the naïve versus axotomy comparison, a 2-fold cut off alone led to an estimated error rate of 16%; combining a >1.5-fold expression change and P < 0.05 significance reduced the estimated error to 5%. The 2-fold cut off identified 178 genes while the combined >1.5-fold and P < 0.05 criteria generated 240 putatively regulated genes, which we have listed. Many of these have not been described as regulated in the DRG by axotomy. Northern blot, quantitative slot blots and in situ hybridization verified the expression of 24 transcripts. These data showed an 83% concordance rate with the arrays; most mismatches represent genes with low expression levels reflecting limits of array sensitivity. A significant correlation was found between actual mRNA differences and relative changes between microarrays (r2 = 0.8567). Temporal patterns of individual genes regulation varied.


CONCLUSIONS
We identify parameters for microarray analysis which reduce error while identifying many putatively regulated genes. Functional classification of these genes suggest reorganization of cell structural components, activation of genes expressed by immune and inflammatory cells and down-regulation of genes involved in neurotransmission.
Costigan
Michael
M
Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA. costigan@helix.mgh.harvard.edu
Befort
Katia
K
Karchewski
Laurie
L
Griffin
Robert S
RS
D'Urso
Donatella
D
Allchorne
Andrew
A
Sitarski
Joanne
J
Mannion
James W
JW
Pratt
Richard E
RE
Woolf
Clifford J
CJ
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D019771:Axotomy; D015152:Blotting, Northern; D003599:Cytoskeleton / Q000235:genetics; D004195:Disease Models, Animal; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome; D017403:In Situ Hybridization; D008297:Male; D009474:Neurons / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D059348:Peripheral Nerve Injuries; D010525:Peripheral Nerves / Q000503:physiopathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D020426:Sciatic Neuropathy / Q000503:physiopathology*; D012680:Sensitivity and Specificity