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Molecular & Cellular Proteomics 6:1574-1588, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples^*,S

R. Hussain Butt^,, Tom A. Pfeifer^,¶,||, Allen Delaney^**, Thomas A. Grigliatti^¶, Wolfram G. Tetzlaff^|| and Jens R. Coorssen^,

From the Department of Physiology and Biophysics, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada, ^¶ Department of Zoology, Life Sciences Center, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada, ^|| International Collaboration of Repair Discoveries, 6270 University Blvd., Vancouver, British Columbia V6T 1Z4, Canada, and ^** British Columbia Cancer Agency (BCCA) Genome Sciences Centre, Vancouver, British Columbia V5Z 1L3, Canada

Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.

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