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Critical factors for successful microarray and real-time PCR analyses

R&D Department
QIAGEN GmbH, Hilden, Germany Gene expression profiling at the RNA level has been significantly facilitated by microarray analysis and quantitative real-time RT-PCR. Microarray analysis offers the advantage of profiling expression levels of hundreds or thousands of genes simultaneously using asingle RNA preparation. Although real-time PCR analysis provides precise quantification over a wider dynamic range of expression levels, it is not suited for simultaneous analysis of large numbers of genes. Therefore, array analysis is often used as a tool to screen for target genes that are differentially expressed between biological samples, and quantitative real-time RT-PCR then provides precise quantification and validation of the microarray results. There are several prerequisites for successful and sensitive array analysis and reliable real-time RT-PCR. Critical steps include the preservation of the RNA expression profile in the biological sample as well as the efficiency of cDNA labeling and cleanup. Newly developed reagents and instrumentation enable increased sensitivity in microarray and real-time RT-PCR analysis. This article gives an overview of these critical factors and new developments. Sample collection and processing affects RNA expression pattern

A crucial step prior to analysis is stabilization of the RNA expression pattern in the biological sample. A biological samples RNA expression profile can dramatically change during collection and processing. This is due not only
to gene induction but also enzymatic degradation of RNA and/or down-regulation
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