Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray.
Use of DNA microarrays in neuroscience have been limited to rough screening purposes, mainly because the reliability and sensitivity of available arrays are not high enough. Because only a few hundred to one thousand genes are usually found to change expression levels in most experiments, we attempted to develop a more quantitative array by the following strategies: 1) limit the genes to those relevant to the experimental system, 2) design oligonucleotide probes of specified molecular properties so that more stringent hybridization conditions can be employed, 3) place six spots per probe on one slide and use multiple normalization genes, and 4) use a new type of gold-coated slide with higher S/N ratio. Genes involved in the construction and functioning of the synapse were selected from the literature as well as experimentally by comparing cerebella from hypothyroid and control mice at postnatal day 15 (P15). Although the number of genes covered was not yet large (172 genes), the custom array "Synaptoarray" thus constructed was capable of detecting +/-20% difference in gene expression ratios. Analysis of the postnatal development of the mouse cerebellum by using Synaptoarray demonstrated a general expression pattern with a peak at P7, followed by a decline at P15 and a partial recovery after P21. P10 clearly marked the end of the initial growth stage and a major transcriptional turning point in this system. This result suggests that such a custom array should be useful in monitoring perturbations to the normal developmental program.
Takahashi
Masaki
M
Department of Chemistry and Biological Science, School of Science and Engineering, Aoyama Gakuin University, Sagamihara, Kanagawa, Japan.
Kondoh
Yasumitsu
Y
Tashiro
Hideo
H
Koibuchi
Noriyuki
N
Kuroda
Yoichiro
Y
Tashiro
Tomoko
T
D000818:Animals; D002531:Cerebellum / Q000254:growth & development*; D017931:DNA Primers; D015870:Gene Expression / Q000502:physiology*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013569:Synapses / Q000502:physiology*
D017931:DNA Primers; D015345:Oligonucleotide Probes