"Genius begins great works; labor alone finishes them."

Joseph Joubert






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General Hospital


Capsule Summary

BioMek machine

The Genomics Core is responsible for the conduct of genome-wide expression analyses on blood T-cell, monocyte, and neutrophil samples obtained from trauma and burn patients. The Core also conducts genome-wide expression analyses on other non-blood tissues (primarily skin, muscle and fat) obtained from the burn patients.

The activities of the Core are divided into two principal components:

  1. routine analyses of samples generated from the PORC site initially using the macroscale techniques (see CSSP Core)
  2. methods development to conduct genome wide expression analysis on ever- reducing quantities of RNA, derived from enriched cell populations obtained by macroscale, and subsequently by microfluidics techniques

The Challenge

The overarching goal of our Program during the initial funding period was to evaluate global changes in whole blood leukocyte gene expression, independent of the changes in leukocyte numbers and differential cell populations that occur in severe trauma.  Such an approach fulfills one of our specific aims focusing on the identification of patterns of gene expression that result from severe injury and are associated with different clinical trajectories.  However, it has become readily apparent in the Program that we would greatly enhance our knowledge about the patient response to trauma or burn injury by determining gene expression patterns from more enriched leukocyte populations, and better understanding the contribution of gene expression by individual leukocyte populations to the total leukocyte population. 

The basic challenges first faced in the Program, including the identification of optimal procedures for the isolation of the total cellular RNA from whole blood and reduction of the amount of required starting material have been met with the development and dissemination of macroscale protocols.  As the Program begins to integrate the cell separation devices described in the CSSP Core in funding Years 8-10, with the nucleic acid isolation microfluidics device developed by the CSSP Core, the new challenge is to obtain sufficient quantities of different T-cell sub-types, monocytes, and neutrophils and to purify the small quantities of RNA from these cells for subsequent microarray analyses from critically ill patients in whom blood sampling volumes are frequently limited.  Existing methodologies are generally inadequate to query the entire human transcriptome with the necessary high-throughput flexibility and sensitivity.

GeneChip pseudoimages
GeneChip Pseudoimages

Our Approach

Based on the significant findings from our first five years of funding, we believe that high- throughput transcriptome analysis can elucidate the cell-cell communication and the differential genomic response of assorted cell types in the circulating blood to predict the patient's outcome in response to a trauma or burn injury and reveal novel mechanistic information regarding the nature, duration and complexity of the immuno-inflammatory response.

In our renewal program, we propose to expand our transcriptome analysis by further characterizing and clarifying the involvement and role of individual signaling pathways and functional modules at the level of the transcriptome. For these enriched cell populations, these genomic changes will be coupled to a more detailed high-throughput proteomic analysis of the same cell population, and a functional proteomic description, based on flow cytometry (performed in the Proteomics Core for T-cells and monocytes). It is our expectation that our detailed genomic studies in combination with the high-throughput proteomics, cell phenotype data, and our epidemiological studies will offer a unique and invaluable resource to the scientific and clinical communities to address some of the fundamental questions regarding the human immuno-inflammatory response to traumatic injury and burn.

Major Functions of this Core

The function of the Genomics Core is two-fold. The primary purpose is the service-oriented function of the Core; that is, to provide high-quality genome-wide expression analyses based on the Affymetrix GeneChip™ technology. The second important purpose of the Core is to facilitate the development and implementation of novel genomic technologies for the benefit of the overall Program. The dual functions of the Genomics Core as a service provision group and technology development/adaptation group are similar to that of the Cell Separation and Sample Preparation (CSSP) Core.

The Program learned early in the initial years of funding that success in generating high-quality genomics data in a large, multi- center, clinical research project depends on minimizing technical variability, preventing human errors, and recognizing at an early stage the impact of methodological or product defects. As a service, the Genomics Core continues to oversee quality control and training in a timely and responsible manner for generation of high-quality genomic data.

Technology development and adaptation includes the development and implementation of new standard operating procedures that will put novel technologies, either developed within the Program or taken from other laboratories, into practice successfully. Methodologies based on new array designs, such as human tiling arrays, are now available to detect novel transcripts in the human genome. In addition, human exon arrays permit the detection and interrogation of alternatively spliced forms of the same gene. The challenge is to integrate new approaches for the labeling and amplification of target cRNA or cDNA for chip hybridization from increasingly fewer cells and smaller quantities of starting RNA. This challenge is significant as we evolve toward microfluidics approaches to isolate ever-enriched and purified leukocyte subpopulations.

Technology development is crucial to realizing the genomics-based vision for translational medicine research. One consideration built into our original study design, and continued in the renewal Program, is the intent to archive sufficient RNA samples from these studies to have available should these future technologies become available. This effort will be made with the consideration that new assays must be robust and highly reproducible, have a large dynamic range, be cost-effective, and practical for both researchers and clinical technicians.



Participants

This Core consists of three genomics centers of excellence at Stanford University, Washington University, and the University of Florida at Gainesville. Dr. Ronald W. Davis is Director of the Genomics Core.

Genome Technology Center at Stanford University under the leadership of Dr. Ronald Davis with Dr. Michael Mindrinos

Injury Genomics Group at Washington University in St. Louis under the leadership of Dr. J. Perren Cobb with Dr. Bernard “Buddy” Brownstein

Laboratory of Inflammation Biology and Surgical Science at the University of Florida in Gainesville under the leadership of Dr. Lyle Moldawer with Dr. Henry Baker (Affymetrix Core)