We describe a procedure for the simultaneous extraction of proteins and nucleic acids from the same experimental sample allowing for direct correlations between genetic, genomic, and proteomic data. This approach, using commercially available column-based nucleic acid extraction kits, requires no hazardous chemicals and is a quick, reliable, and consistent method for concomitant protein extraction. Buffer choice is critical to completely solubilize all proteins in the sample. Proteins solubilized in radioimmunoprecipitation assay (RIPA) buffer did not represent the entire profile when compared with conventionally extracted proteins using the same buffer at the one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) level, however, proteins extracted from the columns and solubilized in a two-dimensional (2-D) electrophoresis lysis buffer showed a similar profile to conventionally extracted proteins when analyzed at both the 1-D and the 2-D level. We further showed that proteins extracted using these methods were compatible with Western blot analysis. This technique provides a simple and effective way to analyze protein and nucleic acids simultaneously from the same sample without affecting yield and quality.

Introduction

Biological complexity emerges from different organizational levels in a highly regulated space-time coordination of processes that involves the participation and orchestrated interaction of DNA, RNA, and proteins between each other and the environment. Fully understanding normal biological processes such as cell differentiation, development and aging, and pathological conditions requires integrated genomic, transcriptional, and proteomic studies (1,2,3), which demand the simultaneous isolation of DNA, RNA, and proteins from the same sample.

Quick and reliable methods that perform simultaneous extraction of DNA, RNA, and proteins from a single sample are ideal for the generation of matched samples that can save time and money and allow for the efficient use of small and precious biological samples. Researchers are increasingly turning away from classic RNA and protein extraction techniques, such as phenol-chloroform separation (4) or time-consuming cesium chloride gradient centrifugation, because of the hazardous chemicals used and that the methods are generally unsuited for routine use in the laboratory. Spin column technology is a simple and quick approach to extracting nucleic acids from small biological samples. Furthermore, most column-based procedures do not require the amount of hazardous chemicals that are used in traditional nucleic acid extraction procedures (5).

Recently, Morse and coworkers (5) discussed the combined extraction of RNA and proteins using RNA spin column-based technology, and Hummon et al. (6) showed an improved method for isolation and solubilization of proteins after TRIZOL extraction of RNA and DNA from the same sample. However, none of these authors did a complete analysis of the proteins obtained at the level of two-dimensional (2-D) electrophoresis to compare the protein profile obtained with conventional methods used in proteomics studies. Here we present a methodology to simultaneously extract RNA/proteins and/or DNA, RNA, and proteins from the same sample using commercially available column-based nucleic acid extraction kits. We further compared the protein profile obtained with some of the methods dedicated to extracting proteins using 2-D electrophoresis, and we show that buffer choice is critical in the efficient extraction of proteins from these kits to allow proteomic studies.

Materials and Methods

Tissue Preparation

All experimental procedures involving human placentas were approved by the University of Newcastle Human Ethics Committee and the Hunter New England Health Human Ethics Committee.

Normal term placentas were collected at John Hunter Hospital, and placental tissues were snap-frozen in liquid nitrogen prior to use. Before protein or nucleic acid extraction, samples were pulverized in liquid nitrogen.

Spin Column Extractions

Thirty or ten milligrams pulverized placental tissue were homogenized in buffer RLT (RNeasy mini kit; Qiagen, Doncaster, VIC, Australia) or buffer QRL1 (DNA/RNA kit; Qiagen), respectively, using the Ultra-Turrax T25 homogenizer (Janke and Kunkel IKA-Labortechnik, Staufen, Germany). Both buffers were supplemented with β-mercaptoethanol as detailed in the manufacturer's instructions.

Purification of Total RNA and Protein Using the RNeasy Mini Kit

RNA was extracted using the RNeasy kit following the manufacturer's instructions (www1.qiagen.com/literature/handbooks). The flow-through from each of the steps was pooled, and from this, proteins were left to precipitate overnight at −20°C.

Purification of DNA, RNA, and Protein Using the DNA/RNA Kit

DNA and RNA were extracted using the DNA/RNA kit following the manufacturer's instructions (www.qiagen.com/literature/handbooks). The flow-through from each of the steps was pooled. The pellet from the QRV1 centrifugation step was resuspended in buffer QRL1 and added to the pooled flow-through. An equal volume of 100% ethanol was added, and the proteins were precipitated overnight at −20°C.

Following extractions, the absorbance at 260 and 280 nm was measured using a NanoDrop ND-1000 spectrophotometer (Biolab, Mulgrave, VIC, Australia), and the ratios were calculated. Nucleic acid quality was further examined by agarose gel electrophoresis.

Protein Extraction

Conventional

Tissues were homogenized on ice using an Ultra-Turrax homogenizer in either radioimmunoprecipitation assay (RIPA) protein extraction buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 1 mM dithiothreitol (DTT), pH 7.4] supplemented with Complete Protease Inhibitor Cocktail tablets (Roche Diagnostics Australia Pty Ltd., Castle Hill, NSW Australia) or 2-D buffer (30 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS). One milliliter buffer was added per 100 mg tissue. The homogenate was then centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected.