Analytical Biochemistry 359 (2006) 274–276 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY 0003-2697/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.07.044 Notes & Tips DiVerential enrichment of high- and low-molecular weight proteins and concurrent RNA extraction David A. MacIntyre, Roger Smith, Eng-Cheng Chan ¤ Mothers and Babies Research Center, University of Newcastle, Newcastle, NSW 2305, Australia Received 6 July 2006 Available online 22 August 2006 Extraction methods often are chosen to maximize the data that may be obtained such as the simultaneous isola- tion of messenger RNA (mRNA) 1 and proteins from a lim- ited amount of tissue. Such methods reduce time, costs, and sampling error. More critically, they facilitate more mean- ingful interpretation and correlation of data. Although sev- eral methods for simultaneous RNA–protein extraction are used [1–3], it remains diYcult to establish whether divergent results are due to sample preparation and handling or underlying biology. We compared two such established methods routinely used interchangeably for simultaneous extraction of RNA and proteins, Trizol LS reagent (Invit- rogen Life Technologies, Carlsbad, CA, USA) and guanidi- nium isothiocyanate (GITC) media [4–7], against a third “protein-only” method using two-dimensional (2D) elec- trophoresis buVer to determine whether similar protein proWles are obtained. Comparisons between Trizol LS and GITC-based RNA extractions have been reported previ- ously [8]; however, a complete lack of information exists regarding the proteins extracted simultaneously using these methods. Both the Trizol LS and GITC methods are modi- Wed versions of the procedure Wrst described by Chomczyn- ski and Sacchi in 1987 [6], and although they use very similar organic solvents and salts, intriguingly diVerent pro- Wles of proteins with very diVerent ranges of molecular sizes are obtained (Fig. 1). Trizol LS preferentially extracts low- molecular weight species, whereas GITC extracts high- molecular weight proteins. These results highlight the importance of using the same analysis protocol for valid comparison and interpretation of results from a sample population. Importantly, the protocols are useful for pref- erential extraction of low- and high-molecular size proteins, respectively. In this study, we compared the protein proWles of human myometrium extracted using three common methods. This tissue can be obtained only from volunteers during major surgery (i.e., Caesarean section); hence, eYcient extraction procedures that will yield maximum data are sought. All experimental procedures used in this study were approved by the University of Newcastle Ethics Committee and the Hunter Area Research Ethics Committee in accordance with the John Hunter Hospital institutional guidelines. Informed consent was obtained from women prior to elec- tive Caesarean sections. Tissue slivers were obtained from the upper margins of the lower uterine segment and were immediately snap-frozen in liquid nitrogen for storage at ¡80 °C until analysis. Frozen myometrium samples (»1 g each) were pulver- ized in liquid nitrogen and stored as frozen aliquots. Equal amounts of the aliquots (100 mg) were homogenized in 1 ml of either (i) Trizol LS reagent; (ii) GITC extraction media consisting of 4 M GITC, 25 mM sodium citrate, 0.5% sarco- syl, and 0.1 M 2-mercaptoethanol; or (iii) 2D electrophore- sis lysis buVer consisting of 5 M urea, 1 M thiourea, 1% Chaps, and 1% Triton X-100 (pH 7). Extractions were repeated at least three times on tissues from Wve women. Proteins were extracted using Trizol LS reagent according to the manufacturer’s instructions. BrieXy, 100 mg of crushed myometrial tissue was homogenized on ice with 1 ml Trizol LS reagent. The supernatant was removed, and to this 0.2 ml chloroform was added and incubated at room temperature for 5 min. The mixture was then centrifuged at 12,000g for 15 min at 4 °C. Following removal of the RNA- containing aqueous phase, DNA was precipitated with eth- anol from the interphase and organic phase and was centri- fuged at 2000g for 5 min at 4 °C. The resulting supernatant * Corresponding author. Fax: +61 2 49214394. E-mail address: cheng.chan@newcastle.edu.au (E.-C. Chan). 1 Abbreviations used: mRNA, messenger RNA; GITC, guanidinium isothiocyanate; 2D, two-dimensional; SDS, sodium dodecyl sulfate; 1D, one-dimensional; PAGE, polyacrylamide gel electrophoresis.