January 15, 2005 (Vol. 25, No. 2)

Technology Offers Automated Separation and Analysis

Gel electrophoresis, which allows separation of DNA and protein samples, is one of the most widely-used techniques in life sciences, but is limited by lack of automation, low reproducibility, and problems in comparing data.

Now there’s an alternativeAgilent Technologies’ (Palo Alto, CA) 5100 Automated Lab-on-a-Chip Platform (5100 ALP) allows fully-automated separation and analysis of nearly 4,000 samples at a time with an analysis-per-sample time that’s ten times faster than standard gel electrophoresis. The development opens the door to streamlined large-scale genomics and proteomics applications.

At the heart of the 5100 ALP is a reusable microfluidic chip for separation and detection with software that allows generation of high quality digital data ready for archiving and data mining. The launch shows how far Agilent Technologies’ life sciences and the chemical analysis business has progressed since the launch of its first commercial lab-on-a-chip system in 1999.

Integrated Biology Approach

“For the last five years, we have been focusing on gene expression,” says Barney Saunders, Ph.D., senior director, life science technology and strategy. “But gene expression alone is not enough to accelerate drug discovery. We found that our customers were, increasingly, wanting to take an integrated systems approach to research, putting together information from DNA, RNA, proteins, and the cell itself.”

This is why Agilent is now embarking on an integrated biology strategy. Recently, it set up a business unit dedicated to developing technology platforms and informatics solutions which will enable researchers to do experiments they previously could not.

“Researchers now want to address key biological questions using more than one form of measurement,” says Dr. Saunders. “In cancer, for example, the arrays we have developed let you look at transcription factors on a genome-wide basis and also at expression signatures. This means being able to take a more effective look at whole pathways in cancer.”

Another important example of an integrated biology approach is comparative genome hybridization (CGH), which can identify multiple and missing chromosomes of segments in a cancer genome. Agilent will introduce an integrated solution for array-based CGH, working with partners such as the Institut Gustave Roussy in Paris, where the technique is being used to develop a more personalized approach to breast cancer, predicting metastasis and treatment response.

The researchers, led by Gilbert Lenoir, are hoping to define a molecular signature of metastasis for melanoma, thyroid, and breast cancers. “We believe the integrated solution for CGH will be a real breakthrough in cancer diagnosis that can be applied to many other genetic diseases,” explains Dr. Saunders.

Another recent development from Agilent is the first tool to measure RNA quality and grade it on a quantitative scale from one to ten. The measurement, known as the RNA integrity number (RIN), will enable new investigations in cancer and cardiovascular therapeutics.

The 5100 ALP allows high quality analysis of PCR fragments for the first time, and Christian Maercker, Ph.D., head of the screening and expression profiling group at the German Resource Center for Genome Research in Heidelberg, Germany, describes how powerful this can be in a range of DNA applications. His lab is currently processing around 800 samples a day and has clear reproducibility of data in projects where quality control is seen as vital.

These include production of cDNA microarrays, looking for the full open-reading frame of the genome, and exploring siRNAs. “The high throughput of the 5100 is much appreciated,” he says. “It saves time, is more reproducible than gel electrophoresis, and is strongly recommended for high throughput analysis of PCR fragments.”

Proteomic Applications

Agilent is also looking at proteomic applications as this market continues to grow. Tony Owen, Ph.D., marketing manager, liquid phase analysis, outlines some of the challenges: Proteins are present in a wide range of concentrations in cells and interesting proteins are present in low abundance.

“Biomarker and protein identification are where we see the best opportunities for the solutions we offer,” he says. For instance, the Multiple Affinity Removal Column allows low abundance, but biologically significant, proteins in a sample to be investigated.

Meanwhile, a number of new platforms related to MS will be introduced during 2005 so more proteins can be identified rapidly and with more confidence.

For proteins, microfluidics developments at Agilent have also led to the HPLC-Chip/MS for liquid chromatography coupled to mass spectrometric analysis, which will be useful in applications such as identifying biomarkers, according to Fred Strohmeier, general manager of the liquid phase analysis business.

The new chip simplifies challenging separations with no need for any connections or fittings. Future prospects in this area include on-chip digestion. “This can remove a major bottleneck for our customers,” promises Strohmeier.

Paul Hawtin, of the global sciences and information department of AstraZeneca (Cheshire, U.K.), is responsible for the supply of purified proteins throughout the company. He describes high throughput protein analysis using the 5100 ALP.

“Probably the most important factor is the availability of qualitative and quantitative data, which has been of great benefit for us.” There have also been advantages in health and safety in not having to use stains and other chemicals, and it is a real bonus not to have to eyeball data because of the electronic data format.

For instance, the 5100 ALP has been used to study an enzyme produced in a baculovirus system in two different cell types. A clear resolution between the two constructs was seen, at a difference of only six amino acids around a thrombin cleavage site. “These are the kinds of things that we might have otherwise missed,” says Hawtin.

The 5100 ALP has also been important in assisting protein re-folding. Some proteins are not expressed in a soluble form and must be re-folded. The platform allows researchers to set up an array to discover the optimum re-folding parameters.

In the next half of the decade, Dr. Saunders believes Agilent can help researchers do what they could not with existing technology. “We will offer tools and informatics solutions that scientists can use to ask deeper and more fundamental biological questions.”

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