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Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

Nature Protocols volume 8, pages 2240–2255 (2013)Cite this article

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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.

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Figure 1: Schematic workflow for obtaining immediate flanking sections and simultaneous extraction of nucleic acids and proteins from the same samples.
Figure 2: Examples of DNA and RNA quality, and integrity from isolated samples.
Figure 3: Gene expression profiling distinguishes tumors from normal samples with high accuracy.
Figure 4: Genome-wide correlation of DNA copy-number alterations with gene expression profiling in tumors.
Figure 5: Correlation of mutations in DNA with loss of protein expression.

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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).

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Authors and Affiliations

  1. Department of Internal Medicine, University of Texas (UT) Southwestern Medical Center, Dallas, Texas, USA

    Samuel Peña-Llopis & James Brugarolas

  2. Department of Developmental Biology, UT Southwestern Medical Center, Dallas, Texas, USA

    Samuel Peña-Llopis & James Brugarolas

  3. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, Texas, USA

    Samuel Peña-Llopis & James Brugarolas

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  1. Samuel Peña-Llopis
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S.P.-L. developed the protocols and performed all the experiments. S.P.-L. and J.B. designed the experiments and wrote the manuscript.

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Correspondence to Samuel Peña-Llopis or James Brugarolas.

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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

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