Biological Aging and Circadian Mechanisms in Murine Brown Adipose Tissue, Inguinal White Adipose Tissue, and Liver (Nov 2009 dataset)

The circadian profile of the transcriptomes in murine tissues was compared between groups of young and old male mice. The oscillatory phase and absolute expression levels were evaluated as a function of biological age. Distinct differences in multiple pathways were apparent based on biological and circadian time in a tissue specific manner. A total of 18 young (5 month) and old (24 month) C57BL/6 mice were acclimated to a set 12 hr light dark cycle and fed ad lib on standard lab chow for 2 weeks in the PBRC Comparative Biology Core Facility. Five days prior to the study, the mice were converted to constant darkness (red light) regimen. On the day of the study, mice were euthanized by CO2 asphyxiation after ad lib feeding overnight in groups of 3 animals per young or old cohort at 4 hr intervals beginning at 7 AM and ending at 3 AM on a single day (July, 2009). The body weight of each animal was recorded prior to dissection of the following tissues: brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and liver. Individual tissues were weighed prior to freezing in liquid nitrogen. Samples were stored at -80oC until use.Total RNA was isolated from the BAT, eWAT, and liver tissues using TriReagent (MRC, Cincinnati OH) in accordance with the manufacturer's recommendations. The Illumina TotalPrep RNA Amplification Kit (Applied Biosystems Inc., Foster City, CA, Catalog #AMIL1791) was used to create labeled cRNA from 750ng of input total RNA according to the manufacturer's protocol. The labeled cRNA samples were then assessed for quality and quantity using a NanoDrop and an Agilent Bioanalyzer. The MouseWG-6 v2 Beadchip (Illumina Sentrix Beadchip Array #11278593) contains 45,200 transcripts and allows 6 samples to be interrogated in parallel. 1.5ug of each labeled cRNA was hybridized to each array according to the manufacturer's protocol. Experimental group samples were distributed randomly across all beadchips. After an 18 hour hybridization at 58°C, the beadchips were processed according to manufacturer's protocol and scanned using an Illumina BeadArray Reader (Illumina, Inc., San Diego, CA).本研究对年轻与老年雄性小鼠各组织的转录组（transcriptome）昼夜节律谱开展对比分析，以生物年龄为自变量，评估节律振荡相位与绝对表达水平。研究发现，多条通路存在显著差异，且该差异具有组织特异性，随生物年龄与昼夜时间呈动态变化。本实验于PBRC比较生物学核心实验室开展，共使用18只C57BL/6品系小鼠，其中年轻组为5月龄，老年组为24月龄。所有小鼠先经12小时固定明暗循环光照适应，并以标准实验室饲料自由采食2周。实验前5天，将小鼠转移至恒暗（红光照明）环境饲养。实验当日（2009年7月），小鼠经整夜自由采食后，采用二氧化碳窒息法实施安乐死。采样以每4小时为一个时间间隔，从当日上午7时至次日凌晨3时，每年轻、老年组每次取3只小鼠。解剖前记录每只小鼠的体重，采集的组织包括棕色脂肪组织（BAT）、腹股沟白色脂肪组织（iWAT）及肝脏。各组织称重后立即置于液氮中速冻，样品保存于-80℃冰箱直至后续实验。采用TriReagent试剂（美国俄亥俄州辛辛那提市MRC公司），按照制造商操作说明从BAT、附睾白色脂肪组织（eWAT）及肝脏组织中提取总RNA。使用Illumina TotalPrep RNA扩增试剂盒（美国加利福尼亚州福斯特城应用生物系统公司，货号AMIL1791），以750ng总RNA为起始模板，按照制造商提供的实验方案合成标记cRNA。随后使用NanoDrop分光光度计与Agilent生物分析仪对标记cRNA样品的质量与浓度进行检测。MouseWG-6 v2微珠芯片（Illumina Sentrix Beadchip Array #11278593）包含45200个转录本，可同时并行检测6个样品。按照制造商实验方案，将1.5μg每份标记cRNA与芯片进行杂交，实验分组样品随机分配至所有微珠芯片中。在58℃杂交18小时后，按照制造商操作流程对微珠芯片进行处理，并使用Illumina BeadArray Reader扫描仪（美国加利福尼亚州圣地亚哥Illumina公司）进行扫描。