Susanne R. Berglund, Chad W. Schwietert, Angela A. Jones, Robin L

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Presentation transcript:

Optimized Methodology for Sequential Extraction of RNA and Protein from Small Human Skin Biopsies Susanne R. Berglund, Chad W. Schwietert, Angela A. Jones, Robin L. Stern, Joerg Lehmann, Zelanna Goldberg Journal of Investigative Dermatology Volume 127, Issue 2, Pages 349-353 (February 2007) DOI: 10.1038/sj.jid.5700557 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 RNA 2100 Bioanalyzer analysis of FastPrep samples. The RNA was run on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) using the RNA 6000 Pico LabChip kit to determine the quality of the samples. The 28S and 18S ribosomal bands show a greater than 2:1 ratio and the calculated RNA ribosomal integrity numbers of the samples ranged from 8.4 to 8.9 verifying high quality RNA. Shown above are the gel images for 11 RNA samples and below are two representative electrophoretic graphs showing the RNA peaks. Journal of Investigative Dermatology 2007 127, 349-353DOI: (10.1038/sj.jid.5700557) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Two-dimensional gel electrophoresis. Protein was isolated from a single 3mm biopsy using our dual extraction method with the FastPrep bead beater. Approximately 20μg of protein was focused on a 11cm, pH 3–10 ReadyStrip IPG gel (Bio-Rad, Hercules, CA). The second dimension was run on a 12.5% polyacrylamide gel and the proteins were silver stained. Journal of Investigative Dermatology 2007 127, 349-353DOI: (10.1038/sj.jid.5700557) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Western blots using biopsy sample protein. Approximately 10–15μg of protein from two different biopsy samples processed with the FastPrep 120 (Q-BIOgene, Irvine, CA) were used to determine the quality of Western blotting. The top panel was probed with mouse anti-cyclin B1 and the bottom panel is mouse anti-GADD 45. The GADD 45 antibody used (Santa Cruz Biotechnology Inc., Santa Cruz, CA) recognizes both the alpha and beta subunits of the protein. Journal of Investigative Dermatology 2007 127, 349-353DOI: (10.1038/sj.jid.5700557) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions