Hepatic gene expression changes following antisense oligonucleotide-based inhibition of miR-29a

Adult BALB/c female mice were injected intraperitoneally with a single dose at 20 mg per kg of antisense oligonucleotide either against miR-29a (5’-TAACCGATTTCAGATGGTGCTA-3’) or against a scrambled sequence (5’-TCATTGGCATGTACCATGCAGCT-3’ Antisense oligonucleotides contained 2’-O-methoxyethyl (2’-MOE), 2’-flouro (2’-F) 2'-alpha-flouro units with a phosphorothioate backbone (Regulus Therapeutics). Six days following the injection, liver was isolated, total RNA was prepared as described above, and the RNA was amplified and biotinylated using the MessageAmp Premier kit (Ambion). Samples (n=4 each experimental and control) were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays in the Children’s Hospital of Philadelphia Nucleic Acids Core Facility and analyzed with the assistance of the Penn Bioinformatics Core. Probe intensities were normalized using the GCRMA method and the significance of the log2-transformed, GCRMA-normalized signal intensities was determined using SAM Adult mice injected with anti-miR-29a or scrambled control ASO, n=4 per group

本实验以成年雌性BALB/c小鼠为对象，通过腹腔注射单剂量20 mg/kg的反义寡核苷酸（antisense oligonucleotide），分别靶向miR-29a（序列为5’-TAACCGATTTCAGATGGTGCTA-3’）或乱序对照序列（序列为5’-TCATTGGCATGTACCATGCAGCT-3’）。所用反义寡核苷酸带有2'-O-甲氧基乙基（2’-MOE）、2'-氟（2’-F）及2'-α-氟修饰，并采用硫代磷酸酯骨架，由Regulus Therapeutics公司提供。给药6天后分离肝脏，按照前述方法提取总RNA，随后使用Ambion公司的MessageAmp Premier试剂盒对RNA进行扩增与生物素标记。实验组与对照组各4个样本，于费城儿童医院核酸核心实验室与Affymetrix GeneChip小鼠基因组430 2.0芯片进行杂交，并在宾夕法尼亚大学生物信息学核心实验室的协助下完成数据分析。采用GCRMA方法对探针信号强度进行标准化处理，对经log2转换且GCRMA标准化后的信号强度，通过SAM方法计算其统计学显著性。本数据集包含注射抗miR-29a反义寡核苷酸或乱序对照反义寡核苷酸的小鼠样本，每组样本量n=4。