Sanjay N Rakhade | PubMed Health

PubMed2005

A common pattern of persistent gene activation in human neocortical epileptic foci.

Epilepsy is a disease of recurrent seizures that can develop after a wide range of brain insults. Although surgical resection of focal regions of seizure onset can result in clinical improvement, the molecular mechanisms that produce and maintain focal hyperexcitability are not understood. Here, we demonstrate a regional, persistent induction of a common group of genes in human epileptic neocortex in 17 patients with neocortical epilepsy, regardless of the underlying pathology. This relatively small group of common genes, identified using complementary DNA microarrays and confirmed with quantitative reverse transcription polymerase chain reaction and immunostaining, include the immediate early gene transcription factors EGR-1, EGR-2, and c-fos, with roles in learning and memory, and signaling genes such as the dual-specificity kinase/phosphatase MKP-3. Maximal expression of these genes was observed in neurons in neocortical layers II through IV. These neurons also showed persistent cyclic adenosine monophosphate response element binding protein (CREB) activation and nuclear translocation of EGR-2 and c-fos proteins. In two patients, local interictal epileptiform discharge frequencies correlated precisely with the expression of these genes, suggesting that these genes either are directly modulated by the degree of epileptic activity or help sustain ongoing epileptic activity. The identification of a common set of genes and the persistent activation of CREB signaling in human epileptic foci provide a clinically relevant set of biological markers with potential importance for developing future diagnostic and therapeutic options in human epilepsy.

Adolescent↗

PubMed2004

Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells.

BACKGROUND: cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells. RESULTS: We performed parallel experiments on identical samples using a dye-swap design with ANOVA and an experimental design that excludes systematic biases by "correcting" experimental/control hybridization ratios with control/control hybridizations on a spot-by-spot basis. We refer to this approach as the "control correction method" (CCM). Using replicate arrays, we identified a decrease in proliferation genes and an increase in differentiation genes. Using an arbitrary cut-off of 1.7-fold and p values <0.05, we identified a total of 32 differentially expressed genes, 9 with the dye-swap method, 18 with the CCM, and 5 genes with both methods. 23 of these 32 genes were subsequently verified by northern blotting. Most of these were <2-fold changes. While the dye-swap method (using either ANOVA or Bayesian analysis) detected a smaller number of genes (14-16) compared to the CCM (46), it was more accurate (89-92% vs. 75%). Compared to the northern blot results, for most genes, the microarray results underestimated the fold change, implicating the importance of detecting these small changes. CONCLUSIONS: We validated two experimental design paradigms for cDNA microarray experiments capable of detecting small (<2-fold) changes in gene expression with excellent fidelity that revealed potentially important genes associated with the anti-proliferative effects of neuregulin on MCF10AT breast epithelial cells.

Blotting, Northern↗