Biological Aging and Circadian Mechanisms in Murine Brown Adipose Tissue, Inguinal White Adipose Tissue, and Liver (Jan 2010 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).本研究比较了年轻与老年雄性小鼠不同组织的转录组昼夜节律特征，以生物学年龄为变量，分析转录组的振荡相位与绝对表达水平。结果显示，不同组织中多条通路的差异表现会随生物学年龄与昼夜时间呈现显著组织特异性变化。 本实验于PBRC比较生物学核心实验室开展，共使用18只C57BL/6小鼠，其中年轻组为5月龄、老年组为24月龄。所有小鼠先适应12小时光照-黑暗循环的光照制度，并自由采食标准实验饲料，饲养周期为2周。实验开始前5天，将小鼠转移至全黑暗（红光照明）环境中饲养。 实验当日，两组小鼠均在夜间自由采食后，于2009年7月的单日中，从上午7时至次日凌晨3时，每隔4小时以二氧化碳窒息法处死，每组每次处死3只小鼠。处死前记录每只小鼠的体重，随后解剖采集棕色脂肪组织（brown adipose tissue, BAT）、腹股沟白色脂肪组织（inguinal white adipose tissue, iWAT）与肝脏组织。单份组织称重后，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（MRC公司，美国俄亥俄州辛辛那提），按照厂商说明书从BAT、eWAT及肝脏组织中提取总RNA。采用Illumina TotalPrep RNA扩增试剂盒（美国应用生物系统公司，加利福尼亚州福斯特城，货号AMIL1791），按照厂商操作流程，以750ng总RNA为起始模板合成标记后的cRNA。随后使用NanoDrop超微量分光光度计与Agilent生物分析仪对标记cRNA样品的浓度与质量进行检测。 本实验使用MouseWG-6 v2微珠芯片（Illumina Sentrix Beadchip Array，货号11278593），该芯片可同时检测45200个转录本，单次可并行分析6个样品。按照厂商操作流程，将1.5μg每份的标记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。于58℃杂交18小时后，按照厂商操作流程对微珠芯片进行处理，并使用Illumina BeadArray阅读器（美国Illumina公司，加利福尼亚州圣地亚哥）进行扫描。