Translational microarray analysis of liver samples treated with PredTox hepatotoxic compound FP014SC

Liver samples were lysed (15 mM Tris-HCl (pH 8.0), 300 mM NaCl and 15 mM MgCl2, plus inhibitors (1 mg/ml heparin 100 µg/ml cycloheximide and 80 U RNAsin)), added to a 10-50% sucrose gradient and ultracentrifuged at 182,000x g for 2 hours. The gradient was aliquotted into 16 1ml fractions and added to Tri reagent (Sigma). RNA was extracted as per the manufacturer's instructions and then sub-pooled into fractions corresponding to monosomes, light polysomes, medium polysomes and heavy polysomes, according to ribosomal density (more ribosomal occupancy = higher translational activity = heavier, and therefore, located in the more dense fractions). Microarray analysis was performed by hybridising control (vehicle treated) monosomes against test (high-dose treated) monosomes on one microarray, control light polysomes against test light polysomes on a second microarray, and so forth for each sub-pool of fractions. Following microarray analysis there were a maximum of four values for each mRNA, corresponding to the proportional representation of that mRNA within each sub-pool of fractions. By calculating the change in values, i.e. degree of slope, across the monosomal region, the light polysomal region, the medium polysomal region and the dense polysomal region it was possible to determine any translational change in activity. In addition, the overall transcriptional change could be analysed for each mRNA. Dual colour microarrays performed on n = 3 samples, implementing a dye swap design. Control samples were treated with vehicle only. The treated samples had 1120mg/kg of the compound (Tetraethyl[(3-hydroxy-2-pyridyl)amino]-methanediphosphonate) for 15 days.

对肝脏样本进行裂解（裂解液成分为15 mM Tris-HCl（pH 8.0）、300 mM NaCl及15 mM MgCl₂，添加抑制剂：1 mg/ml肝素、100 µg/ml环己酰亚胺及80 U RNAsin），随后将裂解液加入10%-50%蔗糖梯度溶液中，以182,000倍重力超速离心2小时。将梯度溶液分馏为16份1 ml的组分，加入Tri试剂（Tri reagent，Sigma）中。按照试剂制造商的说明书提取RNA，随后根据核糖体密度将组分划分为四个亚池：单核糖体（monosomes）组分、轻多核糖体（light polysomes）组分、中等多核糖体（medium polysomes）组分与重多核糖体（heavy polysomes）组分（核糖体占有率越高，翻译活性越强，组分密度越高，对应更重的多核糖体组分）。微阵列（microarray）分析采用如下方式：在第一张微阵列上，将对照（赋形剂处理）单核糖体与处理（高剂量给药）单核糖体进行杂交；在第二张微阵列上，将对照轻多核糖体与处理轻多核糖体进行杂交；其余亚池组分以此类推完成杂交。微阵列分析完成后，每个mRNA最多可获得四个数值，分别对应该mRNA在各亚池组分中的占比。通过计算单核糖体区域、轻多核糖体区域、中等多核糖体区域及重多核糖体区域的数值变化（即斜率），即可确定mRNA的翻译活性变化；此外还可分析每个mRNA的整体转录水平变化。本实验采用n=3个生物学重复样本进行双色微阵列分析，并实施染料交换设计。对照组样本仅接受赋形剂处理；给药组样本则以1120mg/kg剂量的该化合物（四乙基[(3-羟基-2-吡啶基)氨基]甲烷二膦酸盐）处理15天。