The mice were housed in groups of 6 for 1 month and ingested water containing green tea catechin or nothing (control). Every three mice 2 months of age were anesthetized with isoflurane and blood was removed from the jugular vein. The hippocampus was removed and frozen immediately. Total RNA was extracted from the hippocampus using an RNeasy Mini Kit (74104, Qiagen, Valencia, CA, USA). Total RNA was processed to synthesize biotinylated cRNA using One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA, USA) and then hybridized to a Total RNA Mouse Gene 1.0 ST Array (Affymetrix), with 3 biological repeats per group. Raw data were parametrically normalized [50 (link)] by using the SuperNORM data service (Skylight Biotech Inc., Akita, Japan). The significance of GT-catechin ingestion was statistically tested by two-way ANOVA [51 (link)] at p < 0.001.
To compare the effects of GT-catechin ingestion, we performed principal component analysis (PCA) [52 (link)] on ANOVA-positive genes [53 (link)]. To reduce the effects of individual variability among samples, the axes of PCA were estimated on a matrix of each group’s sample means and applied to all data, which were centered using the sample means of control mice.
To compare the effects of GT-catechin ingestion, we performed principal component analysis (PCA) [52 (link)] on ANOVA-positive genes [53 (link)]. To reduce the effects of individual variability among samples, the axes of PCA were estimated on a matrix of each group’s sample means and applied to all data, which were centered using the sample means of control mice.
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