title: Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications.
abstract: Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), mRNA, noncoding RNA (ncRNA; enriched in miRNA) and protein from the same tissues. After tissue selection, about 12-16 extractions of DNA, RNA or protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins.
authors.affiliation: 1] Department of Internal Medicine, University of Texas (UT) Southwestern Medical Center, Dallas, Texas, USA. [2] Department of Developmental Biology, UT Southwestern Medical Center, Dallas, Texas, USA. [3] Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Peña-Llopis
authors.forename: James
authors.initials: J
authors.lastname: Brugarolas
mesh_terms: D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004252:DNA Mutational Analysis; D023281:Genomics / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000302:isolation & purification*; D008867:Microtomy; D016612:Paraffin Embedding; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D035683:MicroRNAs; D012313:RNA; D004247:DNA