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April 01, 2010 (Vol. 30, No. 7)

High-Density Reverse-Phase Protein Arrays

Designing Solutions for Biomarker Research at the Protein Level

  • Click Image To Enlarge +
    Figure 1A. Anti TP53 reactivity with cancer RPPAs. Figure 1B. Quantitative data analysis of anti-ERBB2 antibody response.

    The quest to cure cancer requires an ever-expanding tool kit to identify and validate biomarkers for better detection, diagnosis, prognosis, and treatment of cancer. 

    The vast number of discoveries being made with new technologies have required the development and implementation of comprehensive quantitative methods to link these discoveries with biospecimens from various cancers. Array technologies including DNA microarrays, qPCR arrays, antibody arrays, recombinant protein arrays, tissue arrays, and reverse phase protein arrays (RPPA) are among the methods being used for this task.

    Many of the platforms in use are complementary to each other, but only a few are able to interrogate hundreds or thousands of biospecimens with sufficient sensitivity and deliver relevant quantitative results at an affordable price. Solutions available from OriGene Technologies use qPCR methods at the RNA level and high-density tissue lysate RPPA at the protein level.

    Cancer RPPAs are protein lysates from cancer and normal tissues arrayed at high-density on a nitrocellulose-coated glass slide, which allows simultaneous interrogation of hundreds of samples using high-quality antibodies (Figure 1A). A major use of these arrays is identification and validation of potential biomarkers. This is accomplished by measuring quantitative differences in the expression profiles and post-translational modifications of proteins from cancer and normal tissues (Figures 1B and 2).

  • Developing RPPAs

    Click Image To Enlarge +
    Figure 2. Simple analysis of cancer differential expression index

    High-density RPPAs require a large number of high-quality human biospecimens, which must be banked at reputable institutes, contain a high percentage of tumor, include comprehensive clinical data, and be verified by a certified pathologist. Such samples are often expensive and hard to find.

    OriGene’s cancer biorepository of 12,000 donors allows its researchers to use a large number of samples across 11 cancers for comprehensive cancer RPPAs.

    Another challenge is extracting and standardizing a large number of samples from various tissues and different tumor content. The most straightforward solution is using a single standard operating procedure for all samples, regardless of tumor content.

    Some researchers use laser-capture microdissection to enrich the sample for only cancer cells. Although this method is more accurate, it is often unnecessary and results in limited and expensive material. Mild nondenaturizing detergents (NP40 and Deoxy-cholate) with complete protease and phosphatase inhibitor cocktails are preferred in the extraction process in order to preserve the structure and phosphorylation state of the proteins.

    Quantifying the different lysates remains a problem. The most commonly used method is protein concentration, in which all samples are diluted to a single concentration (usually 1 mg/mL), followed by 4–5 serial dilutions, which provides a single standardized curve. This curve is printed in triplicate for more accurate quantification.

    This procedure is sufficient to accurately detect quantitative changes among samples (Figures 1B and 2). OriGene has found an 84% match between IHC data in the patient pathology report and the expression level measured by the array for ERBB2 among breast samples. These were further confirmed by Western blot analysis.

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