Enabling coupled quantitative genomics and proteomics analyses from rat spinal cord samples.
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.
Butt
R Hussain
RH
Department of Physiology and Biophysics, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Pfeifer
Tom A
TA
Delaney
Allen
A
Grigliatti
Thomas A
TA
Tetzlaff
Wolfram G
WG
Coorssen
Jens R
JR
D000818:Animals; D001331:Automation; D015180:Electrophoresis, Gel, Two-Dimensional; D023281:Genomics / Q000379:methods*; D006146:Guanidines / Q000494:pharmacology; D008297:Male; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D010636:Phenols / Q000494:pharmacology; D014176:Protein Biosynthesis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000378:metabolism*
D006146:Guanidines; D010636:Phenols; D020543:Proteome; C411644:trizol; D012313:RNA