TY - JOUR
T1 - Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications
AU - Peña-Llopis, Samuel
AU - Brugarolas, James
N1 - Funding Information:
acknoWleDGMents We thank S. Vega-Rubín-de-Celis and A. Pavía-Jiménez for critically reviewing the manuscript. This work was supported by a Postdoctoral Fellowship of Excellence from Generalitat Valenciana (Spain) (no. BPOSTDOC06/004) to S.P.-L. and the following grants to J.B.: a grant from the Cancer Prevention and Research Institute of Texas (no. RP101075) and an American Cancer Society Research Scholar grant (no. 55927). J.B. is a Virginia Murchison Linthicum Endowed Scholar in Medical Research. The tissue management shared resource was supported in part by the US National Cancer Institute (no. 1P30CA142543).
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84887005877&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84887005877&partnerID=8YFLogxK
U2 - 10.1038/nprot.2013.141
DO - 10.1038/nprot.2013.141
M3 - Article
C2 - 24136348
AN - SCOPUS:84887005877
SN - 1754-2189
VL - 8
SP - 2240
EP - 2255
JO - Nature Protocols
JF - Nature Protocols
IS - 11
ER -