Time series of diabetes and exercise training induced expression changes in skeletal muscle of mice

Experiment protocol: Experiment was performed on 10 to 15 weeks old male NMRI mice (Harlan, Holland) housed in standard conditions (temperature 22°C, humidity 60 ± 10 %, artificial light from 8.00 am to 8.00 pm, normally 5 animals per cage). Animals had free access to tap water and food pellets (R36, Labfor, Stockholm, Sweden). Animals were randomly divided into healthy and diabetic groups. The diabetic group received a single peritoneal injection of streptozotocin (STZ, Sigma-Aldrich, France, 180 mg/kg) dissolved in sodium citrate buffer solution (0.1 mol/l, pH 4.5) to induce experimental diabetes similar to type 1. The other group received injection of an equal volume of buffer. Diabetes was confirmed 72 hours after the injection by urine glucose testing (Glukotest(r), Roche, Germany), and mice were characterized diabetic when urine glucose values were greater than 200 mg/dl. Diabetic and healthy animals were randomly assigned into 12 groups (n = 5 per group), which were sedentary or trained for one, three or five weeks. Training groups performed 1 hour per day of treadmill running at 21 m/min and 2.5° incline. After one day of familiarization on a rodent treadmill, the mice ran as described above 5 days per week. Mice were sacrificed 24 hours after the last training bout (respective sedentary controls at the same time) by cervical dislocation followed by decapitation. Calf muscles were removed, dissected free of fat and connective tissue, weighed, snap frozen in liquid nitrogen and stored at -80°C for further analysis. Total RNA extraction and sample preparation: Total RNA was extracted from the left calf muscle complex (soleus + gastrocnemius + plantaris) with Trizol Reagent (Invitrogen, Carlsbad, CA) and further purified with RNeasy columns (Qiagen, Valencia, CA) according to the manufacturers' protocols. Concentration and purity of RNA was determined by measuring absorbances at wavelengths 260 and 280 nm. Integrity of the RNA was checked with agarose gel electrophoresis. RNA samples were pooled within each group for microarray analyses. Concentration, purity and integrity of pooled RNA samples were checked as described above. Micro array analysis: Pooled RNA samples were analyzed with Affymetrix Gene Chip MG U74Av2 (Affymetrix , Inc., Santa Clara, CA) representing 6000 known genes and 6000 ESTs. Microarray analyses were performed according to the instructions of Affymetrix. Briefly, 5 µg of total pooled RNA was reverse transcribed using T7-(dT)24-primers and SuperScript II RT enzyme (Invitrogen). Single stranded cDNA was turned to double stranded cDNA using T4 DNA polymerase (Invitrogen). Produced cDNA was then purified and transcribed in vitro to biotin labeled cRNA using Enzo Bioarray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). RNA was purified and its quality was checked. Purity and quantity of RNA was measured with Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Montchanin, DE) and integrity was tested with agarose gel electrophoresis. Sample was then fragmented and hybridized to Affymetrix test chip to check the function of the hybridization cocktail and to ensure adequate representation of both 5´and 3´ends of the RNA. After testing, samples (15 µg) were hybridized to expression chips. After hybridization, chips were washed and stained in Affymetrix Fluidics Station 400. Arrays were stained first with R-Phycoerythrin Streptavidin (Molecular Probes, Eugene, OR), then with biotinylated anti-streptavidin antibody (Vector Laboratories, Burlingame, CA) and again with R-Phycoerythrin Streptavidin for signal enhancement. The chip was scanned with GeneArray Scanner G2500A (Agilent, Palo Alto, CA) Scaling and normalization of data: The array images were analyzed with Microarray Suite 5.0 (Affymetrix) software. All chips were scaled (global scaling) to target intensity of 50 to minimize differences between chips caused by physical differences in chips, hybridization efficiencies and manual laboratory work. The data was subjected to robust normalization that reduces the errors, which are caused by binding capacity and linearity differences between probe sets. Keywords: time-course

实验方案：本实验使用10至15周龄的雄性NMRI小鼠（Harlan，荷兰），饲养于标准环境中：温度22℃，湿度60±10%，人工光照时段为早8:00至晚8:00，每笼饲养5只动物。小鼠可自由获取自来水与饲料颗粒（R36，Labfor，瑞典斯德哥尔摩）。将动物随机分为健康组与糖尿病模型组：糖尿病模型组单次腹腔注射溶于柠檬酸钠缓冲液（0.1mol/L，pH 4.5）的链脲佐菌素（streptozotocin, STZ，Sigma-Aldrich，法国，180mg/kg），以诱导类似1型糖尿病的实验模型；对照组则注射等体积的缓冲液。注射72小时后，通过尿糖检测（Glukotest(r)，罗氏，德国）确认造模成功，尿糖值＞200mg/dl的小鼠被判定为糖尿病造模成功。将糖尿病与健康小鼠随机分为12组（每组n=5），分别为静息组，或接受1周、3周、5周运动训练的组别。训练组每日进行1小时跑台训练，训练速度为21m/min，坡度为2.5°。小鼠先经过1天的啮齿类动物跑台适应训练，之后每周5天按照上述方案进行运动。最后一次训练24小时后，通过颈椎脱臼后断头的方式处死小鼠（同时间点的静息对照组一并处死）。取下小腿肌肉，剥离脂肪与结缔组织后称重，经液氮快速冷冻后保存于-80℃，以待后续分析。
总RNA提取与样本制备：采用Trizol试剂（Invitrogen，加利福尼亚州卡尔斯巴德）从左侧小腿肌肉复合体（比目鱼肌+腓肠肌+跖肌）中提取总RNA，并按照制造商方案使用RNeasy柱（Qiagen，加利福尼亚州瓦伦西亚）进行纯化。通过检测260nm与280nm波长处的吸光度值，确定RNA的浓度与纯度；通过琼脂糖凝胶电泳检测RNA的完整性。将每组内的RNA样本混合后用于芯片分析，混合后的RNA样本再次按照上述方法检测浓度、纯度与完整性。
芯片分析：混合后的RNA样本采用Affymetrix Gene Chip MG U74Av2芯片（Affymetrix公司，加利福尼亚州圣克拉拉）进行分析，该芯片涵盖6000个已知基因与6000个表达序列标签（Expressed Sequence Tags, ESTs）。芯片分析严格遵循Affymetrix的操作指南进行，简要流程如下：取5μg混合总RNA，使用T7-(dT)24引物与SuperScript II逆转录酶（Invitrogen）逆转录合成单链cDNA；随后使用T4 DNA聚合酶（Invitrogen）将单链cDNA转化为双链cDNA。纯化所得双链cDNA后，使用Enzo Bioarray高通量RNA转录标记试剂盒（Enzo Diagnostics，纽约州法明代尔）进行体外转录，合成生物素标记的cRNA。纯化cRNA并检测其质量，通过Nanodrop ND-1000分光光度计（Nanodrop Technologies，特拉华州蒙尚）测定cRNA的纯度与总量，通过琼脂糖凝胶电泳检测其完整性。随后将样本片段化，与Affymetrix测试芯片杂交，以验证杂交液的功能并确保RNA的5'端与3'端均得到充分覆盖。验证完成后，取15μg样本与表达芯片进行杂交。杂交完成后，在Affymetrix Fluidics Station 400中对芯片进行洗涤与染色：首先使用R-藻红蛋白链霉亲和素（Molecular Probes，俄勒冈州尤金）进行染色，随后使用生物素化抗链霉亲和素抗体（Vector Laboratories，加利福尼亚州伯灵格姆），再次使用R-藻红蛋白链霉亲和素以增强信号。最后使用GeneArray Scanner G2500A扫描仪（Agilent，加利福尼亚州帕洛阿尔托）对芯片进行扫描。
数据标化与归一化：使用Microarray Suite 5.0（Affymetrix）软件分析芯片图像。所有芯片均进行全局标化，将目标信号强度统一至50，以最小化由芯片物理差异、杂交效率与实验室操作误差导致的芯片间差异。随后对数据进行稳健归一化，以降低由探针组结合能力与线性差异引发的误差。
关键词：时间序列（time-course）