Expression profiling of the early postnatal stage of Polycystic Kidney Disease in the B6C3Fe a/a-bpck mice

To understand the molecular and cellular mechanisms of pathogenesis of autosomal recessive polycystic kidney disease , we performed a microarray gene expression profiling in early stage kidneys of B6C3Fe a/a-bpck mutant and wild-type mice at postnatal day 3. Genes with over 1.5-fold expression changes in mutant kidneys compared with age matched wild-type tissues were selected for analysis. This study represents the first widespread profiling of B6C3Fe a/a-bpck mutant mouse kidney and provides a valuable platform for better understanding the molecular mechanisms of polycystic kidney disease in human. Mutational analysis was performed in accordance with the protocol of Jackson Laboratory, total RNA was extracted from kidneys of 3 days of postnatal age using a monophasic solution of phenol/guanidine isothiocyanate and TRIzol reagent (Invitrogen) according to their manual, and the samples were incubated with RNase-free DNase I (Ambion). The quality and concentration of each sample were confirmed by spectrophotometry. Affymetrix 430 2.0 arrays were used according to standard Affymetrix procedures. Data analysis was performed with dChip software (Dec.2010 version). These measurements were confirmed by Real-Time PCR.为阐明常染色体隐性遗传性多囊肾病（autosomal recessive polycystic kidney disease）的分子与细胞致病机制，本研究对出生后第3天的B6C3Fe a/a-bpck突变型与野生型小鼠的早期肾脏组织开展了基因芯片表达谱分析。筛选出突变型肾脏相较于年龄匹配的野生型组织表达变化超过1.5倍的基因用于后续分析。本研究首次对B6C3Fe a/a-bpck突变型小鼠肾脏进行了大范围的表达谱分析，为深入解析人类多囊肾病的分子致病机制提供了宝贵的研究平台。本研究严格遵循杰克逊实验室（Jackson Laboratory）的实验方案：从出生后3天的小鼠肾脏中提取总RNA，依照酚/异硫氰酸胍单相裂解液及Invitrogen公司的TRIzol试剂产品手册进行操作，随后使用Ambion公司的无RNase脱氧核糖核酸酶I（RNase-free DNase I）对样本进行孵育处理。通过分光光度法验证了每份样本的质量与浓度。采用Affymetrix 430 2.0基因芯片，严格按照Affymetrix标准流程完成实验。数据分析使用dChip软件（2010年12月版本）进行。本研究的检测结果通过实时荧光定量PCR（Real-Time PCR）得到了验证。