Researchers from Applied Biosystems and the University of Ghent Demonstrate Effectiveness of Megaplex(TM) miRNA Profiling Tools on Small Samples

Researchers from the Ghent University Hospital in Belgium and Applied Biosystems Inc. (NYSE:ABI) have published a study demonstrating the effectiveness of a new method for profiling microRNAs in small samples. The study, entitled "High-throughput stem-loop RT-PCR miRNA expression profiling using minute amounts of input RNA,” describes the simultaneous profiling of 450 human microRNAs from samples as small as a single cell. The paper appears online in the journal Nucleic Acids Research. The study provides validation for a new workflow for miRNA analysis developed by Applied Biosystems to enable highly sensitive, high-throughput detection of miRNAs from extremely small biological samples.

MicroRNAs are a recently discovered class of non-coding small RNA molecules that are receiving attention from the scientific community for their important role in regulating gene expression and for recent research implicating these molecules in complex disease processes such as cancer. Since the discovery of miRNAs, more than 800 human miRNAs have been identified and many more are awaiting experimental validation. In order to harness the potential of this important class of gene regulators, scientists require high-throughput and sensitive detection methods capable of measuring miRNA expression from very small biological samples, including single cells.

The purpose of the study was to test the ability of new tools based on Applied Biosystems’ stem-looped, primer-based real-time PCR approach and industry-standard TaqMan® chemistry to accurately identify miRNAs in three different neuroblastoma cancer cell lines, both with and without a pre-amplification step. In the study, sensitivity to single-cell levels was achieved using a novel approach incorporating an early-access version of the company’s recently commercialized Megaplex™ Primer Pools.

Previous studies have reported the use of pre-amplification in combination with the stem-loop procedure; however, no published study thus far has evaluated the effects of the pre-amplification step on the fidelity of miRNA expression measurement. For that reason, one of the main goals of the study was to evaluate whether pre-amplification would compromise the accuracy of the results.

The researchers in this study reported unbiased and efficient pre-amplification efficiency, irrespective of miRNA expression level, using this method. In fact, pre-amplification actually enabled the detection of miRNAs that were undetectable in the experiments without pre-amplification, despite the use of 40-fold less starting material.

The Megaplex Primer Pools consist of the Megaplex RT Primers and the Megaplex PreAmp Primers. The Megaplex™ RT Primers are a set of highly multiplexed reverse transcription (RT) primers that greatly reduce the number of RT reactions required, thereby streamlining the workflow and reducing the sample input required to create a comprehensive miRNA profile. Megaplex™ PreAmp Primers, used in conjunction with the TaqMan^® PreAmp Master Mix, enable amplification of the starting RNA sample prior to performing detection using real-time PCR, significantly increasing sensitivity when working with limited samples.

"The high specificity and sensitivity of this method makes it ideal for high-throughput miRNA expression profiling,” said Jo Vandesompele, Ph.D., professor at Ghent University Hospital. "This novel technology from Applied Biosystems opens the way for profiling the miRNAome from small cell populations or individual cells.”

Other technologies enable profiling of multiple miRNAs in a single experiment, but such approaches generally require significant amounts of input RNA – 100ng or more – and therefore preclude the use of very small clinical biopsies or analysis of small subsets of cells or single cells. This limits researchers’ abilities to conduct important experiments for diseases such as cancer in which often only minute samples are collected, or originate from archived formalin fixed paraffin embedded samples in which the genetic material may be degraded and RNA yield is relatively low.

"The ability to detect and accurately quantify all known miRNAs from a few cells or even a single cell with these new tools enables researchers to look further and deeper into the important role of miRNAs in living systems,” said Caifu Chen, Ph.D., scientific fellow at Applied Biosystems. "We expect that researchers will now be able to identify novel biomarkers for complex diseases such as cancer, as well as better understand how miRNAs regulate development of a single neuron or embryo.”

The Megaplex miRNA tools used in this study are now commercially available from Applied Biosystems for use with the TaqMan^® MicroRNA Arrays and with individual TaqMan® MicroRNA Assays. Together, these tools are expected to streamline miRNA analysis workflows for life science researchers and provide comprehensive coverage of miRNA expression in small biological samples or single cells.

The study is available online at: http://nar.oxfordjournals.org.

Applied Biosystems is a global leader in the development and commercialization of instrument-based systems, consumables, software, and services for the life-science market. Combined with miRNA isolation kits and analysis products from its Ambion business, the company offers one of the life-science industry’s most comprehensive lines of microRNA analysis tools. The miRNA TaqMan Assays, the miRNA TaqMan Arrays, along with a complete line of TaqMan Array Gene Signature Panels, and more than 1,000,000 individual TaqMan^® Gene Expression Assays are available on the Applied Biosystems website. More information about Applied Biosystems’ complete line of gene expression analysis solutions is available at www.appliedbiosystems.com or at www.allgenes.com.

About Applied Biosystems Inc.

Applied Biosystems Inc. (formerly known as Applera Corporation) is a global leader in the development and marketing of instrument-based systems, consumables, software, and services for academic research, the life science industry and commercial markets. Driven by its employees' belief in the power of science to improve the human condition, the company commercializes innovative technology solutions for DNA, RNA, protein and small molecule analysis. Customers across the disciplines of academic and clinical research, pharmaceutical research and manufacturing, forensic DNA analysis, and agricultural biotechnology use the company’s tools and services to accelerate scientific discovery, improve processes related to drug discovery and development, detect potentially pathogenic microorganisms, and identify individuals based on DNA sources. Applied Biosystems has a comprehensive service and field applications support team for a global installed base of high-performance genetic and protein analysis solutions. Applied Biosystems Inc. is headquartered in Norwalk, CT. On June 12, 2008, Applera Corporation and Invitrogen Corporation (NASDAQ: IVGN) announced that their Boards of Directors had approved a definitive merger agreement under which Invitrogen will acquire all of the outstanding shares of Applied Biosystems stock. The merger is subject to customary closing conditions and is targeted to close in mid-November 2008, subject to receipt of antitrust clearance under the European Council Merger Regulation. Further information regarding the merger is contained in a joint proxy statement/prospectus and supplement that have been mailed to stockholders of the company and Invitrogen. Investors and security holders are urged to read these documents because they contain important information. Information about Applied Biosystems, including reports and other information filed by the company with the Securities and Exchange Commission, is available at http://www.appliedbiosystems.com. All information in this news release is as of the date of the release, and Applied Biosystems does not undertake any duty to update this information unless required by law.

Applied Biosystems Forward Looking Statements

Certain statements in this press release are forward-looking. These may be identified by the use of forward-looking words or phrases such as "should,” "expect,” and "planned,” among others. These forward-looking statements are based on Applied Biosystems’ current expectations. The Private Securities Litigation Reform Act of 1995 provides a "safe harbor” for such forward-looking statements. In order to comply with the terms of the safe harbor, Applied Biosystems notes that a variety of factors could cause actual results and experience to differ materially from the anticipated results or other expectations expressed in such forward-looking statements. These factors include but are not limited to: (1) rapidly changing technology and dependence on the development and customer acceptance of new products; (2) sales dependent on customers’ spending policies; (3) other factors that might be described from time to time in Applied Biosystems’ filings with the Securities and Exchange Commission.

For Research Use Only. Not for use in diagnostic procedures. Practice of the patented 5’ Nuclease Process requires a license from Applied Biosystems. The purchase of the TaqMan Array Human MicroArray Panel includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser's own internal research when used with the separate purchase of an Authorized 5’ Nuclease Core Kit. No other patent rights are conveyed expressly, by implication, or by estoppel. For further information on purchasing licenses contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. The TaqMan® Low Density Array is covered by U.S. Patents Nos. 6,514,750 and 6,942,837. Micro Fluidic Card developed in collaboration with 3M Company.

©Copyright 2008. Applied Biosystems Inc. All rights reserved. Applied Biosystems, and AB (Design) are registered trademarks and Megaplex is a trademark of Applied Biosystems Inc. or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are trademarks of their respective owners.