Deciphering the Pathways of Disease

In the diagnosis and treatment of life-threatening diseases, early detection often means a better prognosis for the patient. Prostate cancer is an important example. For men, it's the most common type of cancer and the second leading cause of cancer death after lung cancer. It's difficult to conquer if discovered too late but generally curable if diagnosed and treated at an early stage.

Non-invasive testing methods such as prostate-specific antigen (PSA) play a key role in early detection, but the results are sometimes inconclusive. For example, PSA levels are abnormally high in many prostate cancer patients, but nearly half of patients have normal PSA readings. To help provide greater certainty in the detection, classification and monitoring of prostate cancer, researchers are pursuing more-reliable diagnostic markers.

Isolating markers and mapping pathways

With cancer and many other diseases, deciphering the connection between marker and disease starts with gene expression research. When scientists try to isolate diagnostic markers or map disease pathways, four methods often come into play: RNA isolation; gene expression analysis; protein expression; and protein purification.

RNA isolation: Early on, a large number of candidate genes are screened using tools such as microarrays to identify genes of interest. This process starts with the isolation of RNA. Although RNA is thermodynamically stable, it degrades readily in the presence of RNases, and quality control of RNA is critical for obtaining meaningful gene expression data. The Agilent 2100 bioanalyzer with the RNA 6000 Nano LabChip^® kit is the industry standard for quality control of labeled and non-labeled microarray samples.

Example of PCR-product analysis using a simulated gel view produced by the Agilent 2100 bioanalyzer.

Gene expression analysis: Microarray experiments may reveal a subset of genes that are differentially expressed in the context of the study. These genes are then validated using reverse transcription polymerase chain reaction (RT-PCR) methods. When performed in multiplex mode, RT-PCR is an especially precise, accurate way to measure the expression of multiple gene targets. The Agilent 2100 bioanalyzer and DNA LabChip kits allow precise measurement of size and concentration of PCR and RT-PCR products. As an example, researchers at City of Hope National Medical Center in Duarte, California, are using this solution in their prostate cancer research.

Protein expression: After using microarray and RT-PCR experiments to identify a gene of interest, the next step is to express the protein of diseased cells and learn more about their biological function. Using the Agilent 2100 bioanalyzer with the cell assay extension and the Cell Fluorescence LabChip kit, it's possible to verify transfection efficiency and intra-cellular or cell-surface protein expression at the cellular level. The data from simple, on-chip flow cytometric analysis are useful for downstream experiments, protein function research and other cell biology investigations.

Protein purification: In many cases, it's necessary to purify the expressed protein to study its function, structure, substrate bindings and interaction with other proteins. Using the Protein 200 Plus LabChip kit with the Agilent 2100 bioanalyzer enables researchers to monitor and optimize each step of the purification process. Applications include analysis of column fractions for protein purity and concentration determination, detection of specific protein binding with cell lysate flow-through comparison, determination of column capacity to prevent column overloading, optimization of purification conditions to obtain the highest protein purity and yield, and tracking of the protein of interest through the entire purification process.

Whether the goal of disease research is a diagnostic marker, a better therapeutic or a way to achieve outright prevention, Agilent's array of analysis tools can accelerate the pace of discovery and development.

For more information

The Agilent 2100 bioanalyzer and LabChip kits provide a powerful platform for many facets of disease research. To learn more about these and other Agilent life sciences products and resources, please visit the main page of the Life Sciences/Chemical Analysis section of our Web site.