Diated transport and transcription regulators. These genes were characterized by an

Diated transport and transcription Neuromedin N regulators. These genes were characterized by an initial decrease in expression, which persisted even after 6hrs then gradually returned to baseline levels by 12hrs. A few genes seemed to increase by 12hrs, however the increases were small and very close to the untreated control. Genes such as syntaxin 18, Rab8b showed functional overlap for protein transport and localization. Quantitative RT-PCR validation of potential candidates genes We carried out validation of select candidate genes using quantitative PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850275 RT-PCR to assess changes in levels of gene expression. We selected genes for qPCR validation based on high fold change increase or decrease, substantial characterization in previous literature as well as gene expression changes associated with cellular processes that were enriched in the gene clusters. We hypothesized that these processes could increase vulnerability of neurons to degeneration if impaired. We validated three representative genes from clusters showing significant downregulation of expression upon BDNF withdrawal and three genes from clusters that reflected significant BCTC upregulation. Validated gene targets showed expression patterns that were similar to the microarray expression profiles. We found significant changes in the gene coding for the extracellular matrix component Acan. In the microarray profiling, Acan increased by 0.5 fold after 1.5hrs of BDNF withdrawal peaking to 2 fold by 3hrs. By 6hrs Acan expression was decreasing, returning to baseline by 12hrs. A similar profile of change was Dev Neurobiol. Author manuscript; available in PMC 2016 February 01. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Mariga et al. Page 6 seen in the qPCR validation; however Acan increased more than 10-fold as early as 1.5hrs and by 3hrs its expression had already begun to decline although it was still more than 4 fold above control. At 6hrs, expression was still approximately 4 fold relative to control then declined to baseline by 12hrs. Also from cluster 2, striking changes were observed in Dual-Specificity Phosphatases 5, a MAP kinase phosphatase which dephosphorylates Erk1/2. Following BDNF withdrawal, DUSP5 increased 2-fold as early as 1.5hrs, peaked at 3hrs to a 2.3-fold increase then returned to baseline by 612hrs. This trend was reproduced by qPCR validation where DUSP5 increased 4-fold at 1.5hrs, peaked to 6-fold increase at 3hrs then returned to the same levels as control by 12hrs. Another gene that had consistent changes for the microarray and qPCR validation was Sprouty homolog 2; an inhibitor of the MAP kinase pathway. Spry2 expression increased 0.6-fold after 1.5hrs of BDNF withdrawal then gradually declined throughout the timecourse, returning to baseline by 12hrs. qPCR validation also showed Spry2 reproducing a similar profile of change with a 0.6 fold increase at 1.5hrs and corresponding gradual decline from 3hrs to 12hrs. Downregulated genes were selected from cluster 3 for qPCR validation. Golga5, a gene coding for a protein that is important for golgi structure maintenance showed a reproducible decrease in expression both by microarray and qPCR. Upon BDNF withdrawal, Golga5 expression decreased 0.4-fold then remained below baseline with another significant decrease at 612hrs. In the qCR validation, the profile of change is similar, however expression returns to baseline by 12hrs. Rab8b, a Rab-GTPase transport regulator, also decreased 0.3-fold at 36.Diated transport and transcription regulators. These genes were characterized by an initial decrease in expression, which persisted even after 6hrs then gradually returned to baseline levels by 12hrs. A few genes seemed to increase by 12hrs, however the increases were small and very close to the untreated control. Genes such as syntaxin 18, Rab8b showed functional overlap for protein transport and localization. Quantitative RT-PCR validation of potential candidates genes We carried out validation of select candidate genes using quantitative PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850275 RT-PCR to assess changes in levels of gene expression. We selected genes for qPCR validation based on high fold change increase or decrease, substantial characterization in previous literature as well as gene expression changes associated with cellular processes that were enriched in the gene clusters. We hypothesized that these processes could increase vulnerability of neurons to degeneration if impaired. We validated three representative genes from clusters showing significant downregulation of expression upon BDNF withdrawal and three genes from clusters that reflected significant upregulation. Validated gene targets showed expression patterns that were similar to the microarray expression profiles. We found significant changes in the gene coding for the extracellular matrix component Acan. In the microarray profiling, Acan increased by 0.5 fold after 1.5hrs of BDNF withdrawal peaking to 2 fold by 3hrs. By 6hrs Acan expression was decreasing, returning to baseline by 12hrs. A similar profile of change was Dev Neurobiol. Author manuscript; available in PMC 2016 February 01. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Mariga et al. Page 6 seen in the qPCR validation; however Acan increased more than 10-fold as early as 1.5hrs and by 3hrs its expression had already begun to decline although it was still more than 4 fold above control. At 6hrs, expression was still approximately 4 fold relative to control then declined to baseline by 12hrs. Also from cluster 2, striking changes were observed in Dual-Specificity Phosphatases 5, a MAP kinase phosphatase which dephosphorylates Erk1/2. Following BDNF withdrawal, DUSP5 increased 2-fold as early as 1.5hrs, peaked at 3hrs to a 2.3-fold increase then returned to baseline by 612hrs. This trend was reproduced by qPCR validation where DUSP5 increased 4-fold at 1.5hrs, peaked to 6-fold increase at 3hrs then returned to the same levels as control by 12hrs. Another gene that had consistent changes for the microarray and qPCR validation was Sprouty homolog 2; an inhibitor of the MAP kinase pathway. Spry2 expression increased 0.6-fold after 1.5hrs of BDNF withdrawal then gradually declined throughout the timecourse, returning to baseline by 12hrs. qPCR validation also showed Spry2 reproducing a similar profile of change with a 0.6 fold increase at 1.5hrs and corresponding gradual decline from 3hrs to 12hrs. Downregulated genes were selected from cluster 3 for qPCR validation. Golga5, a gene coding for a protein that is important for golgi structure maintenance showed a reproducible decrease in expression both by microarray and qPCR. Upon BDNF withdrawal, Golga5 expression decreased 0.4-fold then remained below baseline with another significant decrease at 612hrs. In the qCR validation, the profile of change is similar, however expression returns to baseline by 12hrs. Rab8b, a Rab-GTPase transport regulator, also decreased 0.3-fold at 36.