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September 01, 2010 (Vol. 30, No. 15)

Efficient Detection and Analysis of miRNAs

Innovative Tools for Screening Hundreds of miRNAs and Subsequent Functional Studies

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    Figure 1. Example of an RT2 miRNA PCR Array assay panel that is specific for 88 cancer-related human miRNAs: The profile shows the expression changes in colon cancer in comparison to normal tissue.

    MiRNAs (microRNAs) are 20–23 nucleotide small RNA molecules that play an important role in post-transcriptional gene regulation in a wide variety of biological processes including development, disease, and apoptosis.

    Scientists must screen hundreds of miRNAs to identify those involved in their pathway or disease of interest. This can be achieved using PCR arrays that allow quantification of hundreds of miRNAs in one experiment. Once screened, further analysis can be performed to verify the results, perform more in-depth functional studies, or get a better understanding of miRNA biogenesis.

    RT2 miRNA PCR Arrays from Qiagen are assays in a 96- or 384-well plate format. Each well of these arrays contains a specific and sensitive primer for one miRNA. This allows high-throughput quantification and profiling of multiple miRNAs in a single RT-PCR run. Data from different disease states, development stages, or tissue types can be compared to identify up- or downregulated molecules.

    RT2 miRNA PCR Arrays target the entire miRNome or subsets of miRNAs that are involved in biological pathways, processes such as inflammation and cell differentiation, or diseases such as cancer. The assays can distinguish between miRNA family members with single nucleotide mismatches as well as differentiate mature miRNAs from their precursors.

    Screening of hundreds of miRNAs can be performed in a single experiment. As the first step, the miRNAs are converted to cDNA by reverse transcription and universal tailing. The master mix with the components for the RT-PCR reaction is then mixed with the sample and added into the well. After mixing, the PCR run is performed in a real-time PCR instrument and data is collected.

    Integrated, web-based software performs gene-expression analysis from uploaded raw data and delivers results in multiple formats, enabling easy interpretation. Differences between different states can be calculated from raw CT values normalized to a panel of housekeeping small nuclear RNAs.

    Quantification of the expression of multiple miRNAs in a cell or tissue type under defined conditions provides an miRNA profile or a snapshot of miRNA expression that can be used to elucidate the role of miRNAs in regulatory pathways or their misregulation in disease. A profile for colon cancer is shown in Figure 1. In this example, many of the miRNAs profiled appear to be upregulated in cancer cells.

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