Expression profiling of human neuroblastoma cells SK-N-SH after treatment with low dose of 2,3,7,8- tetrachlorodibenzo-p-dioxin. Homo sapiens

Exposure to dioxins has been associated with impaired development of the nervous system and losses of brain functions. However, the precise cellular events and related molecular basis underlining these adverse effects has not been fully understood. We aimed to reveal global alterations of mRNA expression and potential cellular events by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Overall design: Messenger RNA microarray analysis was employed on cultured human neuroblastoma cells SK-N-SH exposed to 10-10 M TCDD.Total RNA was extracted from cell samples using Trizol/Chloroform method, and then purified with magnetic beads of Agencourt Ampure (Beckman Coulter, Brea, CA, USA). Target preparation for microarray processing was carried out according to the GeneChip® WT PLUS Reagent Kit. In each preparation, 500 ng total RNA was used for a double-round of cDNA synthesis. After fragmentation of 2nd-cycle single-stranded cDNA, sample was labeled with biotin by terminal deoxynucleotidyl transferase (TdT). Then sample was hybridized to a GeneChip® Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix) with 44699 annotated genes.

二噁英暴露已被证实与神经系统发育受损及脑功能减退相关。然而，介导此类不良效应的确切细胞事件及相关分子机制尚未完全阐明。本研究旨在揭示2,3,7,8-四氯二苯并对二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD）暴露后mRNA表达的全局改变及潜在细胞事件。
实验设计：本研究采用mRNA微阵列分析，对暴露于10^-10 M TCDD的培养人神经母细胞瘤细胞SK-N-SH进行检测。使用Trizol/氯仿法从细胞样本中提取总RNA，随后利用Agencourt Ampure磁珠（贝克曼库尔特，美国加利福尼亚州布雷亚市）进行纯化。按照GeneChip® WT PLUS试剂盒操作流程完成微阵列检测所需的样本靶标制备，每批制备均使用500 ng总RNA开展两轮cDNA合成。第二轮循环的单链cDNA片段化完成后，利用末端脱氧核苷酸转移酶（terminal deoxynucleotidyl transferase, TdT）对样本进行生物素标记。随后将标记后的样本与搭载44699个注释基因的GeneChip®人类转录组阵列2.0（HTA 2.0，Affymetrix）进行杂交。