Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies.
A small amount of bioptic tissue ( approximately 5-10mg 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+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg 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 10mg 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.
Barbero
Giovanna
G
Dipartimento di Genetica, Biologia, Biochimica-Università degli Studi di Torino, 10126 Torino, Italy. giovanna.barbero@unito.it
Carta
Franco
F
Giribaldi
Giuliana
G
Mandili
Giorgia
G
Crobu
Salvatore
S
Ceruti
Carlo
C
Fontana
Dario
D
Destefanis
Paolo
P
Turrini
Francesco
F
D002292:Carcinoma, Renal Cell / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D006801:Humans; D007680:Kidney Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D040901:Proteomics; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009363:Neoplasm Proteins; D012334:RNA, Neoplasm