title: A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol.
abstract: The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at -80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction.
authors: V. G. Vaze College, Mulund, Mumbai, India.
authors: Neah
authors: N
authors: Likhite
authors: Ujjwala M
authors: UM
authors: Warawdekar
mesh_terms: D000199:Actins / Q000032:analysis; D015415:Biomarkers; D015153:Blotting, Western / Q000379:methods; D001943:Breast Neoplasms / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism; D045744:Cell Line, Tumor; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006146:Guanidines / Q000737:chemistry*; D056945:Hep G2 Cells; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D010636:Phenols / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D013048:Specimen Handling; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D000199:Actins; D015415:Biomarkers; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D006146:Guanidines; D017879:Isothiocyanates; D009363:Neoplasm Proteins; D010636:Phenols; D020543:Proteome; D012334:RNA, Neoplasm; D000076105:X-ray Repair Cross Complementing Protein 1; C054435:guanidine isothiocyanate; C411644:trizol