Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples

Abstract

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.

An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

“…Owing to very limited PBMC collected, these were directly solubilised in 2DE buffer containing 8 M urea, 2 M thiourea, 4 % (w/v) CHAPS and a cocktail of protease, kinase and phosphatase inhibitors [14][15][16][17].…”

“…Automated frozen disruption (AFD) of cortex, skeletal muscle and spleen samples, subsequent isolation of total membrane (MP) and soluble protein (SP) fractions and determination of protein concentrations were carried out as previously described [10,[14][15][16][17][18][19]. Owing to very limited PBMC collected, these were directly solubilised in 2DE buffer containing 8 M urea, 2 M thiourea, 4 % (w/v) CHAPS and a cocktail of protease, kinase and phosphatase inhibitors [14][15][16][17].…”

“…isolated MP and SP fractions) were resolved using an established 2DE protocol [10,[15][16][17][18]20]; triplicate gels were resolved for each fraction. As insufficient protein was recovered from these initial PBMC isolates, only a single gel was resolved for the cuprizone treatment and duplicate gels for the parallel controls; as this was the first analysis to test concerns of broader toxicity, we thought it nonetheless prudent to also collect the PBMC data here for further confirmation in more targeted future assessments.…”

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…At the protein level, although newer technologies for resolution and identification are regularly introduced, polyacrylamide gel electrophoresis (PAGE) remains the most accepted, widespread, and successfully implemented technique for the quantitative, high-resolution separation, and characterization of these critical molecules [5]. Utilization of PAGE as a single (native or detergent based PAGE) or second dimension of separation following isoelectric focusing (2D PAGE), has been shown to deliver very high resolution and reproducibility for protein separation [3, [6][7][8][9][10][11][12]. In a single analysis, 2D electrophoresis (2DE) provides information on protein charge, abundance, localization, isoforms, and posttranslational modifications.…”

Use of membrane separation device for extraction of intracellular protein and DNA

“…Currently, the methods of protein or DNA extraction mainly include phase separation and precipitation using guanidinium salts with organic solvents [9,10] and using extraction kits [11]. Some lysis devices and approaches at the microchip level often consume much time on the design and fabrication, and lack of the process of cell washing.…”