Ciprofibrate stimulated gene expression in rats

Samples and RNA preparation: The series is composed of 4 hybridizations for analysis of differentially expressed genes in the liver of ciprofibrate dosed vs. control rats. Rats (200-250 g) were given (by gastric intubation) tap water (control group, n=5) or ciprofibrate (50 mg/kg body weight, once daily for 60 days, n=4). Liver total RNA extraction was performed first by ultracentrifugation on a cesium chloride cushion and then using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: One microgram of total RNA from the control pool (n=5), and from each ciprofibrate dosed rat was reverse transcribed and labeled with Cy3- and Cy5-attached dendrimer, respectively, using 3DNA Array Detection Submicro kit as described in the manufacturer's protocols (Genisphere, Montvale, NJ). The labeling reactions were also spiked with exogenous control RNAs (Stratagene) at expected Cy5/Cy3 ratios of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 0.5, 0.33, 0.25 and 0.125 for control spike 1, 2, 3, 4, 5, 6, 8, 9, 10 and 7, respectively. The Cy3 and Cy5 labeled samples (combined volume 5 microliter) were mixed with 30 microliter of the hybridization solution containing 0.25 M NaPO4, 1 mM EDTA, 4.5% SDS, 1 x SSC, 2 x Denhardt's solution, 0.25 microliter antifade reagent (Genisphere) and 0.2 microgram mouse COT-1 DNA (GIBCO BRL Life Technologies), added onto the array and covered with 22 x 50mm cover slip. The array was assembled into humidified hybridization chamber (Corning) and hybridized at 65oC for 18 h by submerging in a water bath. Post-hybridization washes were done once in 2 x SSC, 0.2% SDS at 55oC for 15 min, then in 2 x SSC at room temperature for 15 min, and finally with 0.2 x SSC at room temperature for 15 min. Scanning, image analysis and data processing: Arrays were scanned at 10 micrometer resolution with a confocal laser scanner constructed in-house in collaboration with NEMKO (Trondheim, Norway) according to a prototype developed at NHGRI (http://www.nhgri.gov/DIR/LCG/15K(HTML/). Microarray image analysis was done with MicroArray Suite software version 2.1 with globally normalization (Scanalytics, Inc., Fairfax VA). First, spots undetected (target intensity=1) in at least one array were excluded. Then, spots with signal intensity (averages of the 4 arrays) less than 300 in both channels were removed. The fluorescence intensity ratios (Cy5/Cy3) for genes measured from probes spotted in duplicate were averaged for each slide. Then, mean ratios of the 4 hybridizations were calculated for each gene, and differentially regulated genes were determined using 1.4 fold change cutoff values (1.4 for up-regulated or 0.7 for down-regulated genes). Keywords: other

样本与RNA制备：本系列实验包含4组杂交反应，用于分析环丙贝特（ciprofibrate）给药组与对照组大鼠肝脏组织中的差异表达基因。选取体重200~250g的大鼠，通过胃插管法分别给予自来水（对照组，n=5）或环丙贝特（50mg/kg体重，每日1次，连续给药60天，n=4）。肝脏总RNA提取先采用氯化铯缓冲液超速离心法，随后使用TRIZOL试剂（TRIZOL Reagent，GIBCO BRL Life Technologies，纽约州纽约市）完成提取。

标记与杂交：分别取对照组混合样本（n=5）及每只环丙贝特给药大鼠的总RNA各1μg，采用3DNA Array Detection Submicro试剂盒（3DNA Array Detection Submicro kit），按照制造商说明书（Genisphere，新泽西州蒙特维尔市）的操作流程，分别使用结合Cy3和Cy5的树状大分子进行反转录标记。标记反应体系中还加入了外源对照RNA（Stratagene），针对编号1至10的对照加样参照，其预设Cy5/Cy3比值分别为：对照spike 1~6依次为1.0、2.0、3.0、4.0、5.0、6.0，对照spike7为0.125，对照spike8~10依次为0.5、0.33、0.25。将Cy3与Cy5标记的样本（总体积5μL）与30μL杂交液混合，该杂交液包含0.25M磷酸钠、1mM EDTA、4.5% SDS、1×SSC、2×Denhardt溶液（Denhardt's solution）、0.25μL抗淬灭试剂（Genisphere）以及0.2μg小鼠COT-1 DNA（COT-1 DNA，GIBCO BRL Life Technologies），将混合液滴加至芯片表面并覆盖22×50mm盖玻片。将芯片组装至加湿杂交舱（Corning）中，浸没于水浴锅内，于65℃杂交18小时。

杂交后洗涤：依次采用如下洗涤步骤：首先在55℃下于2×SSC、0.2% SDS溶液中洗涤15分钟；随后在室温下于2×SSC溶液中洗涤15分钟；最后在室温下于0.2×SSC溶液中洗涤15分钟。

扫描、图像分析与数据处理：采用与挪威特隆赫姆市NEMKO合作研发的共聚焦激光扫描仪，以10μm分辨率对芯片进行扫描，该扫描仪基于美国国立人类基因组研究所（NHGRI）开发的原型机制作（http://www.nhgri.gov/DIR/LCG/15K(HTML/)）。芯片图像分析采用MicroArray Suite软件2.1版本进行全局归一化处理（Scanalytics公司，弗吉尼亚州费尔法克斯市）。首先剔除至少在一张芯片中信号未被检测到（目标强度=1）的点；随后剔除两个通道信号强度均值（4张芯片的平均）均低于300的点。对于以重复点样方式制备的探针所对应的基因，计算每张芯片的荧光强度比值（Cy5/Cy3）的平均值；进而计算4组杂交实验中每个基因的平均比值，并以1.4倍变化作为筛选阈值（上调基因阈值为1.4，下调基因阈值为0.7），以此鉴定差异调控基因。

关键词：其他