Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis

被引：11

作者：

Grzendowski, Michael ^{[1
]}

Riemenschneider, Markus J. ^{[2
]}

Hawranke, Eva ^{[1
]}

Stefanski, Anja ^{[1
]}

Meyer, Helmut E. ^{[1
]}

Reifenberger, Guido ^{[2
]}

Stuehler, Kai ^{[1
]}

机构：

[1] Ruhr Univ Bochum, Med Proteom Ctr, D-44801 Bochum, Germany

[2] Univ Dusseldorf, Inst Neuropathol, Dusseldorf, Germany

来源：

PROTEOMICS | 2009年 / 9卷 / 21期

关键词：

2-D DIGE; Brain tumor; Cesium chloride ultracentrifugation; Sample preparation; Technology; Ultrafiltration; HUMAN-MALIGNANT GLIOMAS; GENE; AMPLIFICATION; EXPRESSION; OVEREXPRESSION; SUBSET; CDK4; RNA;

D O I：

10.1002/pmic.200800902

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high-molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high-salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2-D DIGE, MS and Western blotting.

引用

页码：4985 / 4990

页数：6

共 14 条

[1] SIMULTANEOUS ISOLATION OF DNA, RNA, AND ANTIGENIC PROTEIN EXHIBITING KINASE-ACTIVITY FROM SMALL TUMOR SAMPLES USING GUANIDINE ISOTHIOCYANATE [J].

COOMBS, LM ;

PIGOTT, D ;

PROCTOR, A ;

EYDMANN, M ;

DENNER, J ;

KNOWLES, MA .

ANALYTICAL BIOCHEMISTRY, 1990, 188 (02) :338-343

[2]

GRZENCLOWSKI M, 2009, NEUROONCOLO IN PRESS

[3]

Ichimura K, 1996, ONCOGENE, V13, P1065

[4]

Louis DN., 2007, WHO CLASSIFICATION T

[5]

REIFENBERGER G, 1994, CANCER RES, V54, P4299

[6]

REIFENBERGER G, 1993, CANCER RES, V53, P2736

[7] Frequent biallelic inactivation and transcriptional silencing of the DIRAS3 gene at 1p31 in oligodendroglial tumors with 1p loss [J].

Riemenschneider, Markus J. ;

Reifenberger, Julia ;

Reifenberger, Guido .

INTERNATIONAL JOURNAL OF CANCER, 2008, 122 (11) :2503-2510

[8]

Riemenschneider MJ, 1999, CANCER RES, V59, P6091

[9] Molecular classification of human gliomas using matrix-based comparative genomic hybridization [J].

Roerig, P ;

Nessling, M ;

Radlwimmer, B ;

Joos, S ;

Wrobel, G ;

Schwaenen, C ;

Reifenberger, G ;

Lichter, P .

INTERNATIONAL JOURNAL OF CANCER, 2005, 117 (01) :95-103

[10] Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma [J].

Sitek, B ;

Lüttges, J ;

Marcus, K ;

Klöppel, G ;

Schmiegel, W ;

Meyer, HE ;

Hahn, SA ;

Stühler, K .

PROTEOMICS, 2005, 5 (10) :2665-2679

← 1 2 →