【摘要】 基因芯片是大规模表达谱分析的有力工具 ,有助于阐明疾病发生的分子机制及发现新的诊治靶标。但常规方法需要大量RNA ,每张芯片需要 5 0～ 2 0 0 μg总RNA ,2～ 5 μgmRNA。许多珍贵难得样本都不能满足这一要求 ,成为限制芯片广泛应用的瓶颈。结合模板转移效应 ,优化了基于T7的RNA线性扩增技术 ,可从 3μg以下总RNA得到足量的反义RNA ,克服了这一难题。同一RNA样本的自身比较试验结果显示反义RNA标记的芯片与总RNA、mRNA标记的芯片假阳性率相似。同一对RNA样本的表达谱分析也显示反义RNA标记的芯片与总RNA、mRNA标记的芯片无明显差异。更多还原

【Abstract】 cDNA mircoarrays are powerful parallel tools for gene expression profiling analysis, which help us to understand the molecular mechanism of diseases and to identify potential targets for therapeutic intervention. However, their broader application are hampered by the large amount of RNA required: up to 200 μg of total RNA or 5 μg of mRNA for one chip, making analysis of small samples difficult. In this work, combined with a template switching effect, the T7 RNA linear amplification procedure was optimized, providing multiple copies of anti-sense RNA with reduced sample inputs: no more than 3 μg total RNA for one chip. Using the same RNA sample in all cases, the anti-sense RNA labeling method was compared with standard total RNA and mRNA methods by two sets of self-comparison experiments. Furthermore, the three methods in profiling analysis were compared with the same pair RNA samples. All the results indicated that the fidelity, reproducibility, and reliability showed no significant difference with conventional total RNA or mRNA microarrays.更多还原