The simultaneous extraction of high-molecular-weight DNA and of RNA from solid tumors

doi:10.1016/0003-2697(83)90299-3

Analytical Biochemistry

Volume 134, Issue 2, 15 October 1983, Pages 288-294

https://doi.org/10.1016/0003-2697(83)90299-3 Get rights and content

Abstract

A novel method for the isolation of both macromolecular DNA and RNA from solid tissues based upon the disruption by vibration of deep-frozen material in a mechanical device termed Mikro-dismembrator, is described. This technique reveals a yield of, on the average, 1 to 3 mg of either DNA or RNA per gram of tissue. The quality of the purified nucleic acids permits the detailed analysis of integrated tumor virus DNA sequences and their mRNA transcripts. Furthermore, the efficient isolation of papilloma virions from keratinized wart tissue is facilitated by the application of the Mikro-dismembrator.

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AFD has been described as optimal for the extraction of protein, RNA/DNA, carbohydrates, and lipids when directly compared with competing homogenization strategies (18, 19, 21, 22, 33). This is especially true of tough, fibrous, or otherwise difficult to homogenize biological material (18, 20, 22). From a proteomics standpoint, the benefits of AFD include (but are not limited to) enabling the detection of proteins that are undetectable by other classical homogenization strategies (18).
Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.
European Organisation for Research and Treatment of Cancer (EORTC) Pathobiology Group standard operating procedure for the preparation of human tumour tissue extracts suited for the quantitative analysis of tissue-associated biomarkers
2007, European Journal of Cancer
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If the shaking flask and the sample are not cold enough, the disintegration procedure will fail and the result will be an amorphous mass of partly disintegrated tissue. The shaking flask bead mill technology is already in use and has been applied by several centres for the extraction of RNA, DNA,46–56 or biomarker proteins from various sources of human and animal tissues.57–68 In the following, guidelines are given on how to store tumour tissue (see Fig. 1) and how to process it for the extraction of RNA, DNA, or protein.
With the new concept of ‘individualized treatment and targeted therapies’, tumour tissue-associated biomarkers have been given a new role in selection of cancer patients for treatment and in cancer patient management. Tumour biomarkers can give support to cancer patient stratification and risk assessment, treatment response identification, or to identifying those patients who are expected to respond to certain anticancer drugs. As the field of tumour-associated biomarkers has expanded rapidly over the last years, it has become increasingly apparent that a strong need exists to establish guidelines on how to easily disintegrate the tumour tissue for assessment of the presence of tumour tissue-associated biomarkers. Several mechanical tissue (cell) disruption techniques exist, ranging from bead mill homogenisation and freeze-fracturing through to blade or pestle-type homogenisation, to grinding and ultrasonics. Still, only a few directives have been given on how fresh-frozen tumour tissues should be processed for the extraction and determination of tumour biomarkers. The PathoBiology Group of the European Organisation for Research and Treatment of Cancer therefore has devised a standard operating procedure for the standardised preparation of human tumour tissue extracts which is designed for the quantitative analysis of tumour tissue-associated biomarkers. The easy to follow technical steps involved require 50–300 mg of deep-frozen cancer tissue placed into small size (1.2 ml) cryogenic tubes. These are placed into the shaking flask of a Mikro-Dismembrator S machine (bead mill) to pulverise the tumour tissue in the capped tubes in the deep-frozen state by use of a stainless steel ball, all within 30 s of exposure. RNA is isolated from the pulverised tissue following standard procedures. Proteins are extracted from the still frozen pulverised tissue by addition of Tris-buffered saline to obtain the cytosol fraction of the tumour or by the Tris buffer supplemented with the non-ionic detergent Triton X-100, and, after high-speed centrifugation, are found in the tissue supernatant. The resulting tissue cell debris sediment is a rich source of genomic DNA.
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In the framework of a preliminary study on the transdermal penetration of cyproterone acetate (CPA), a simple and rapid procedure involving an extraction step coupled to a HPLC-UV determination has been developed for the separation and quantification of CPA in the two main skin layers-epidermis and dermis-after local application. The separation of epidermis and dermis layers was carefully carried out by means of a sharp spatula after skin immersion in heated water at 65 °C. The two skin layers were then treated separately according to the same process: (1) sample homogenization by vibration after freezing with liquid nitrogen in a Mikro-Dismembrator^®; (2) CPA extraction with methanol after addition of the internal standard (betamethasone dipropionate); (3) centrifugation; (4) evaporation of a supernatant aliquot; (5) dissolution of the dry residue in methanol and addition of water; (6) centrifugation; (7) injection of a supernatant aliquot into the HPLC system. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of acetonitrile and water (40:60 (v/v)). The method was then validated using a new approach based on accuracy profiles over a CPA concentration range from 33 to 667 ng/ml for each skin layer. Finally, the method was successfully applied to the determination of CPA to several skin samples after topical application of different gel formulations containing CPA.
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Methods: Transporter mRNA and protein levels were assessed by reverse transcription polymerase chain reaction and Western blot analysis. Tissue distribution of transporters was investigated by immunohistochemistry and immunofluorescence microscopy. Hepatic bile acids were measured by gas chromatography-mass spectrometry.
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Conclusions: Down-regulation of basolateral uptake systems and maintenance/up-regulation of canalicular and basolateral efflux pumps may represent adaptive mechanisms limiting the accumulation of toxic biliary constituents.

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The simultaneous extraction of high-molecular-weight DNA and of RNA from solid tumors

Abstract

Anal. Biochem

Virology

Biochem. Biophys. Res. Commun

Eur. J. Biochem