Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies

被引:16
作者
Barbero, G [1 ]
Carta, F
Giribaldi, G
Mandili, G
Crobu, S
Ceruti, C
Fontana, D
Destefanis, P
Turrini, F
机构
[1] Univ Turin, Dipartimento Genet Biol Biochim, I-10126 Turin, Italy
[2] Porto Conte Ric, I-07100 Sassari, Italy
[3] CERMS, Ctr Ric Med Sperimentale, I-10126 Turin, Italy
[4] Dipartimento Discipline Med Chirurg AO Molinette, I-10126 Turin, Italy
关键词
biopsy; 2-DE; kidney; MALDI-TOF; Q-TOF; real-time PCR;
D O I
10.1016/j.ab.2005.10.020
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A small amount of bioptic tissue (similar to 5-10 mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28 +/- 0.05 mg of proteins and 2.5 +/- 0.33 mu g of RNA for each 10 mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10 mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis. (c) 2005 Elsevier Inc. All rights reserved.
引用
收藏
页码:62 / 71
页数:10
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