2University of California, Berkeley, Berkeley, CA, USA
A systems approach is being applied in many areas of the biological sciences, particularly in cancer research. The coordinated, simultaneous extraction of DNA, RNA, and proteins from a single sample is crucial for accurate correlations between genomic aberrations and their consequences on the transcriptome and proteome. We present an approach to extract and completely solubilize up to 98% of the total protein recovered from archived samples following TRIZOL® isolation of RNA and DNA. We also demonstrate using polyacrylamide gel electrophoresis (PAGE) and Western blot analysis that the proteins, representing both a wide molecular weight range and some posttranslational modifications, such as protein phosphorylation, remain stable in phenol-ethanol for up to 3 years at −20°C.
Extraction of DNA, RNA, and proteins from biological samples is a common procedure in molecular biology laboratories for analysis of the genome, transcriptome, and proteome, respectively. TRIZOL®, a monophasic solution of phenol and guanidine isothiocyanate, is designed as a one-stop reagent for the extraction of RNA, DNA, and proteins from tissues or cells (1). While there are numerous publications documenting its utility for the extraction of nucleic acids, fewer reports describe its application to the extraction of proteins. This is mainly due to difficulties in resolubilizing the protein fraction; therefore, the more common procedure is to divide the sample and treat one portion with TRIZOL reagent for RNA and DNA extraction and subject the second portion to a lysis buffer for recovery of the proteins. However, when dealing with small samples such as tumor biopsies, a single extraction reagent is crucial in order to obtain enough material for subsequent analyses. The additional advantage is that all analyses can be performed on the same cell mass; this facilitates direct comparisons of alterations in the genome, transcriptome, and proteome. In this report, we present a method to efficiently extract and solubilize proteins from tissue samples using TRIZOL reagent subsequent to the sequential extraction of RNA and DNA. Additionally, we provide evidence that the proteins and some posttranslational modifications remain stable in phenol-ethanol for up to 3 years at −20°C.Materials and Methods Samples
HCT 116 cells were obtained from ATCC (accession no. CCL-247; Manassas, VA, USA). Cells were cultured in T75 flasks to about 80% confluence in McCoy's 5A Media (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Invitrogen). Prior to harvesting, cells were rinsed with phosphate-buffered saline (PBS). For cells harvested with TRIZOL (Invitrogen), 8 mL TRIZOL were added to each flask, the cells were removed with a cell scraper, and the suspension transferred to a polypropylene tube capable of withstanding high-speed centrifugation [maximum relative centrifugal force (RCF) of 7500× g]. Sequential RNA and DNA extraction was performed as described in the Invitrogen product information (www.invitrogen.com/content/sfs/manuals/15596026.pdf). Protein pellets isolated from phenol-ethanol supernatants were dissolved in 200 µL solvent. In the cases where two solvents were used, 100 µL each solvent were used.
For proteins isolated with a lysis buffer, cells were rinsed with PBS, then 2 mL trypsin (Invitrogen) were added. After a 3-min incubation at 37°C, the cells were rinsed from the flask in 10 mL media containing 10% FBS, transferred to a 15-mL conical tube, and centrifuged and pelleted at 250× g for 10 min. The media was aspirated, and the cells were resuspended and rinsed in PBS. Cells were pelleted again and placed on ice. The PBS was aspirated, and the pellet resuspended in 100 µL TNE lysis buffer [0.25 mL 50 mM Tris, pH 7.5, 40 µL 2 mM EDTA, 87.7 mg NaCL, 22.3 mg Na4P2O7, 2.1 mg NaF, 0.2 mg Na3VO4, 100 µL 1% Nonidet® P40 (NP40), and 9.61 mL water, sterile filtered with a 0.2-µm filter, and stored at 4°C]. Since Na4P2O7, NaF, and Na3VO4 function as phosphatase inhibitors, they were added to each solvent in the same relative amounts as in the TNE lysis buffer. One Complete™ Mini Protease Inhibitor Cocktail Tablet (Roche Applied Science, Indianapolis, IN, USA) was added for every 10 mL TNE lysis buffer. Cells were lysed for 30 min on ice. Each sample described represents the proteins from one harvested T75 flask.
Protein concentrations were determined using the BCA™ Protein Assay Reagent kit (Pierce, Rockford, IL, USA) and fraction V, protease-free bovine serum albumin (BSA; Roche Applied Science) as the protein standard. For nonaqueous protein solutions, a 1:50 dilution in water was measured, and the corresponding concentration calculated.
Tumor biopsies were collected and immediately stored in RNAlater® (Ambion, Austin, TX, USA). Sequential RNA and DNA extraction was done with TRIZOL following the manufacturer's protocol. While evaluations of the RNA by microarray and reverse transcription PCR (RT-PCR) analysis and the DNA by conventional chromosome comparative genomic hybridization (CGH) were successfully performed, the protein fractions were stored at −20°C for 3 years as phenol-ethanol supernatants awaiting subsequent analysis (2,3).Dialysis of Phenol-Ethanol Supernatants
The phenol-ethanol supernatants were loaded into Spectra/Por® 6 regenerated cellulose (RC) dialysis membranes (MWCO 2000; Spectrum Laboratories, Rancho Dominguez, CA, USA) and dialyzed against three changes of an aqueous 0.1% sodium dodecyl sulfate (SDS) solution at 4°C, changing the solution first after 16 h, then after 4 h, and again after 2 h, respectively. For every 1 mL phenol-ethanol supernatant, 100 mL 0.1% SDS solution were used. During dialysis, the samples partitioned into three phases: (i) a colorless supernatant (approximately 85% volume), (ii) a globular mass (approximately 10% volume), and (iii) a colorless, viscous liquid (approximately 5% volume).