Optimization of T7-based RNA Amplification System for cDNA Microarray

LI Tao1, LI Yao*, HAN Zhi-Yong¹, CHEN Qin¹, QIU Min-Yan², NI Sheng², JIANG Zhen-Ling¹, XIE Yi, MAO Yu-Min*

( State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China;
¹ Shanghai BioStar Genechip Inc., Shanghai 200092, China;
² United Gene Holdings, LTD., Shanghai 200092, China )

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 mg of total RNA or 5 mg 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 mg 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.

Key words microarray; RNA linear amplification

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