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. Access through your

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BEING VIEWED BY OTHERS SCALABLE CO-SEQUENCING OF RNA AND DNA FROM INDIVIDUAL NUCLEI Article 12 February 2025 RELIABLE DETECTION OF SOMATIC MUTATIONS IN SOLID TISSUES BY LASER-CAPTURE

MICRODISSECTION AND LOW-INPUT DNA SEQUENCING Article 14 December 2020 COMPREHENSIVE RNA DATASET OF TISSUE AND PLASMA FROM PATIENTS WITH ESOPHAGEAL CANCER OR PRECURSOR LESIONS Article Open

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Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS 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). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Internal

Medicine, University of Texas (UT) Southwestern Medical Center, Dallas, Texas, USA Samuel Peña-Llopis & James Brugarolas * Department of Developmental Biology, UT Southwestern Medical

Center, Dallas, Texas, USA Samuel Peña-Llopis & James Brugarolas * Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, Texas, USA Samuel Peña-Llopis & James

Brugarolas Authors * Samuel Peña-Llopis View author publications You can also search for this author inPubMed Google Scholar * James Brugarolas View author publications You can also search

for this author inPubMed Google Scholar CONTRIBUTIONS S.P.-L. developed the protocols and performed all the experiments. S.P.-L. and J.B. designed the experiments and wrote the manuscript.

CORRESPONDING AUTHORS Correspondence to Samuel Peña-Llopis or James Brugarolas. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Peña-Llopis, S., Brugarolas, J. Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for

genomic applications. _Nat Protoc_ 8, 2240–2255 (2013). https://doi.org/10.1038/nprot.2013.141 Download citation * Published: 17 October 2013 * Issue Date: November 2013 * DOI:

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