Correction
18 Jun 2015: Yamaguchi Y, Li Z, Zhu X, Liu C, Zhang D, et al. (2015) Correction: Polyethylene Oxide (PEO) and Polyethylene Glycol (PEG) Polymer Sieving Matrix for RNA Capillary Electrophoresis. PLOS ONE 10(6): e0131265. https://doi.org/10.1371/journal.pone.0131265 View correction
Figures
Abstract
The selection of sieving polymer for RNA fragments separation by capillary electrophoresis is imperative. We investigated the separation of RNA fragments ranged from 100 to 10,000 nt in polyethylene glycol (PEG) and polyethylene oxide (PEO) solutions with different molecular weight and different concentration. We found that the separation performance of the small RNA fragments (<1000 nt) was improved with the increase of polymer concentration, whereas the separation performance for the large ones (>4000 nt) deteriorated in PEG/PEO solutions when the concentration was above 1.0%/0.6%, respectively. By double logarithmic plot of mobility and RNA fragment size, we revealed three migration regimes for RNA in PEG (300-500k) and PEO (4,000k). Moreover, we calculated the smallest resolvable nucleotide length (Nmin) from the resolution length analysis.
Citation: Yamaguchi Y, Li Z, Zhu X, Liu C, Zhang D, Dou X (2015) Polyethylene Oxide (PEO) and Polyethylene Glycol (PEG) Polymer Sieving Matrix for RNA Capillary Electrophoresis. PLoS ONE 10(5): e0123406. https://doi.org/10.1371/journal.pone.0123406
Academic Editor: Vipul Bansal, School of Applied Sciences—RMIT University, AUSTRALIA
Received: October 29, 2014; Accepted: February 18, 2015; Published: May 1, 2015
Copyright: © 2015 Yamaguchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This project was partly supported by Grant-in-Aid for Scientific Research (Houga) (No. 25600049 (Y.Y.)), JSPS, Japan. The authors also gratefully acknowledge support from National Natural Science Foundation of China (No. 21205078 and No.61378060), Research Fund for the Doctoral Program of Higher Education of China (No. 20123120110002), and the Innovation Fund Project for Graduate Student of Shanghai (No. JWCXSL1401), China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Capillary electrophoresis(CE) is a powerful technology for nucleic acids separation.[1] For the separation of DNA/RNA fragments, especially the longer ones, CE filled with polymer solutions is the only available method for high-throughput analysis. Although microchip has been utilized for DNA sequencing, CE is still a standard and fundamental analytical instrument for DNA sequencing, besides CE is simple and low cost.[2,3] Especially when CE is combined with fluorescence detection method, a widely employed technology in bioanalytical science[4,5,6,7], it will be of great sensitivity. Capillary polymer electrophoresis, in which the polymer solutions were filled in capillary tube, still needs the fundamental investigations, including the direction of high voltage, polymer solutions, buffer solutions,[8,9] and migration process[10,11,12] for the application of hyphenated biological applications[13,14,15], and for RNA fragment separation.
The ratio of charge to nucleic base is uniform for both DNA and RNA. Thus in CE, the separation of nucleic acids requires the sieving matrix to differentiate the migration time. The polymer solution, which is usually the linear polymer solution, is used as the sieving matrix for DNA/RNA separation. When the nucleic acid migrates through the polymer solution, it entangles with the polymer chains. As different size of nucleic acid molecule has the different interaction of entanglement with polymer chains, the migration time is differentiated according to the length of nucleic acid size. Thus the migration process, which is dominated by the entanglement process between the polymer chains and DNA/RNA fragments, is different according to the sample size.[12,16,17]
DNA migration process in electrophoresis has been theoretically and experimentally studied very well in the past few years. [18,19,20,21,22,23,24] In the theory of DNA migration in polymer solutions, there are several regimes for DNA migration process.[20,21,22,25] Each regime is resulted from the migration morphology of the DNA during electrophoresis. The separation regime is also strongly depended on the physical properties of the sieving polymer, such as concentration, polymer length, and polymer chemical compositions.
The migration processes in polymer matrixes are also strongly decided by the properties of the analyte. RNA and DNA are physically deferent molecules and also have many similar properties. Because the sieving process of the DNA/RNA in polymer solution dominates the migration process of them during CE, the stiffness of the DNA or RNA are important physical properties for understanding the DNA/RNA separation in CE. Since the persistence length of DNA is about 20 nm while RNA is about 3 nm,[26,27] the sieving process of the RNA is different from the sieving process of DNA in CE. The approach to further increase the high throughput of RNA analysis is the identification of the sieving matrixes. For polymer solutions, several physical characteristics, including the high-separation performance, excellent chemical stability to buffer solution, low background for fluorescence detection, ease of preparing the polymer solutions, are considered for a high performance and excellent reproducibility of CE.
Polyethylene glycol (PEG) and polyethylene oxide (PEO) were well characterized by the studies of physical[28,29], micelle formation[30], rheological phenomena[31], solution dynamics [32] and morphology[33]. PEO was always employed for DNA sequencing by filling into capillary tubes[34]. PEG/PEO were also employed for the DNA sequencing because the PEO solution possessed homogeneous structure. They were easy to prepare, and were able to provide high resolution power for DNA separation in both direct current (DC) capillary electrophoresis and alternative current (AC) capillary electrophoresis. [35]
In this paper, we demonstrated the separation of RNA (100–10,000 nt) CE to investigate the separation performance of RNA. The sieving polymers were PEG and PEO with different molecular weight: 300–500 k and 4,000 k. We found that the resolution length of RNA was ranging from 6.0 nt to 3522.8 nt and the best length resolution was 6.0 nt in the solution of PEO at 0.8% (w/w).
Material and Methods
Chemicals
PEG of average molecular weight (Mw) of 4 k in powder was purchased from Sigma-Aldrich, Co. (Atlanta, USA). PEG(300–500 k) and PEO (4,000 k) powders were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). The polymer solutions were prepared by adding the polymer powder, TBE powder (TAKARA, Shanghai, China) and urea powder (Sinopharm Chemical Reagnent Co., Ltd, Shanghai, China) into ultrapure water, and then the solutions were stirred for more than 12 hours.[35] Then the homogenous polymer solutions were degased using a vacuum pump. Finally, the polymer solutions were diluted to different concentration and applied to the CE system. The RNA marker (TAKARA BIO INC., Dailian, China) were diluted to a solution of 350 ng/μl concentration, which also contained 4.0 M urea and 0.5× TBE. Prior to CE, RNA sample was denatured by 4.0 M at 65°C for 5.0 min, and then was fast cooled on ice for 3.0 min. SYBR Green II were bought from Invitrogen (Carlsbad, CA, USA).
Capillary Electrophoresis
The CE system had been described in previous works.[36] The total length and effective length of the 75μm ID fused-silica capillary (Polymicro, Yamato, USA) were 9 cm and 6 cm, respectively. It was coated by S. Hjertén method to suppress the electroosmotic flow (EOF).[37] A high-voltage power supply was employed to provide high voltage between the two ends of the capillary. The exciting light from a mercury lamp was filtered by an optical filter (U-MWIB3, Olympus, Japan) to a wavelength of 460–495 nm, which was the excitation wavelength of the conjugate of SYBR Green II and the nucleic acid. The excited fluorescence emission was collected by a 100× objective (MPlanFL N, Olympus). A photomultiplier tube (H8249-101, Hamamatsu Photonics, Japan) was used to detect the collected light. LabVIEW software (National Instrument, Austin, TX, USA) was applied for high-voltage power supply control and data collection. After each run, the capillary was flushed with ultrapure water by a vacuum pump. The whole CE detection proceeded in a dark box under room temperature.
Resolution and resolution length (RSL) of the peaks
The resolution (Rs)[38] was determined by the following equation: Rs = 2 Δx / (w1 + w2). Here, Δx is the difference of the migration time of the concessive two peaks in electropherogram and w is the time of the full width of maximum of the peak (w1) and following peak (w2). The Gaussian curve fitting was performed by Origin Pro 8 SR0. The author’s observation was applied to find the width where the RNA peaks in electrophoretogram with considering the tailing, especially the peaks corresponding to the RNA longer than 1,000 nt, consistently appearing in the all experiment.
The resolution length (RSL) was determined by the following equation: RSL = Δn / Rs, where Δn is the RNA fragments length difference of adjacent peaks in electrophoretogram and Rs is the resolution. RSL is for the determination of the minimum RNA nucleotide length, which are the base pairs that can be separated with baseline separation in which the resolution is equal to 1.
Results and Discussion
The fundamental structures of PEG and PEO are the same, because both of them are the polymers of ethylene oxide. The polymer of ethylene oxide with the molecular weight above 20,000g/mol is PEO and the one with molecular weight below 20,000 g/mol is PEG. We prepared PEO (4,000 k) and PEG (300–500 k), PEG(4 k) for our experiment, and observed the cognizable peaks, which correspond to the RNA ladders migration in the above polymer solutions.
The purpose of the investigation of the RNA migration in PEG/PEO solution is to find the regimes of the RNA sieving process in these polymer solutions and the quality of the separation, which is quantified by the minimum resolvable difference of the nucleotide length. PEG(4 k) was tested as the sieving matrix for CE first, but no recognizable peak appeared in the electrophoretogram when PEG polymer concentrations were 5%,10%, 15%, and 20%.
We then performed the separation of RNA in PEG(300–500 k) with various concentrations, and the polymer solution contained 4.0 M urea. The RNA sample was denatured by 4.0 M urea prior to injection. Data in Fig 1 demonstrated that RNA ranging in size from 100 nt to 3,000 nt was baseline resolved when the polymer concentrations were 1.0% and 1.2%, but the longer RNA (> 5000 nt) can hardly be observed, which was possibly because the volume of them was too little. For diluted PEG solutions (0.6% and 0.8%), RNA sample shorter than 4,000 nt could be separated within 7.0 min, although the resolution for the adjacent RNA fragments was not better than they were in the concentrated solutions. For the more diluted PEG solutions (<4.0%), the peaks in the electropherogram were overlapped, and thus they cannot be resolved. We also attempted the RNA separation in the more concentrated PEG solutions (2.4% and 3.6%), but found that no recognized peaks appeared in the electropherogram. Moreover, compared with the RNA migration in hydroxyethyl cellulose (HEC) solution[10,39,40], it seems that RNA moves much faster in PEO solution than it is in HEC solution.
Capillary electrophoresis was performed at 100V/cm. The total length and the effective length of the capillary were 9.0 cm and 6.0 cm, respectively. Sample Loadings: 100 V/cm (2.0 sec).
We repeated the same experiment for RNA separation in PEO(4,000 k) solutions by CE, and the results were demonstrated in Fig 2. It showed that all 14 RNA fragments were resolved in 0.4% PEG solution, this was most possibly because the length of the polymer in PEO (4,000 k) was longer than that of PEG (300–500 k). When the concentration of PEO increased from 0.4% to 1.0%, only RNA fragments from 100 nt to 4.000 nt were baseline resolved. Comparing the separation performance of RNA in PEG(300–500 k) with that in PEO(4,000 k), the long RNA fragments (4,000, 5,000, 6,000 and 10,000 nt) were separated only in 0.4% and 0.2% PEO solutions, which has a higher molecular weight, indicating that PEO solution yielded better solution for longer RNA fragments. RNA fragments from 100 nt to 1,000 nt were also well separated in PEO solutions concentrated from 0.4% to 1.0%. This was because the radii of gyration of the RNA molecules had the same order of magnitude with the pore size of the polymer matrix. [41,42] Thus we can concluded that the higher concentration of PEO solution served the higher performance for the shorter RNA, and the lower concentration of PEO solution favored longer RNA. We also attempted the RNA separation in 1.5% and 2.0% PEO polymer solution, but we observed only slight peaks of 100–1,000 nt RNA fragments in 1.5% PEO solution and no separated peaks in 2.0% PEO solution (not showed in the Figures). This might because the pore size of the network in these concentrated polymer solutions was too small to allow the RNA fragments get through.
Sample and capillary electrophorrtic conditions were the same as those in Fig 1.
The migration patterns of RNA in PEG (300–500 k) solution was evaluated by double logarithmic plot of the RNA length and mobility (Fig 3). Three regimes were observed. They were Ogston (section I), reptation regime (section II) and reptation with orientation (section III), and were consistent with DNA migration in polymer solution. [43] The threshold of the regime was around 300 nt to 700 nt which was approximately 90 nm. Because the radius of gyration of PEO polymer was estimated at about 40 nm, the point of the changing the model was justified.[41] And in the experiment of 4,000 k PEO solution, the same plot (Fig 4) was prepared to find the regimes. Three regimes were found from the plot and those were corresponding to Ogstion, reptation, reptation with orientation.
The plot was prepared based on the electrophoresis in Fig 1.
The plot was prepared based on the electrophoresis in Fig 2.
The separation performance of RNA was also evaluated by the resolution length (Fig 5). Resolution length tells the minimum, which is smallest RNA nucleotides length resolvable with baseline separation. Theoretically, two adjacent RNA fragments with 1 nt difference in size could be resolved if the resolution length is equal to 1. The resolution length of RNA in PEG(300–500 k) was tabulated in Table 1. It showed that the resolution length varied from 12.7 nt to 761.4 nt, and the best, minimum resoluble nt was 12.7 nt for RNA between 100 nt and 200 nt. (Fig 5A) It was greatly improved than the resolution length (30.2 nt) observed in HEC solution by pulsed filed CE[39]. Because the minimum number (Nmin) was the nucleotide when the resolution was 1, the resolution 0.02 was required for 1 nucleotide deference separation. The resolution length for RNA in PEO(4000 k) was tabulated in Table 2, and it demonstrated that the resolution length (Table 2) varied from 6.0 nt to 3522.8 nt, where the best, minimum resoluble nucleotide length was 6.0 nt between 100 nt to 200 nt. (Fig 5B) For the separation of RNA fragments below 1,000 nt the PEO with 4,000 k showed better performance. Furthermore, we can find from Table 1 and Table 2 that the threshold in the migration regime moves to the short RNA fragments when the concentration of the polymer increases.
(a) 300k-500k PEG polymer solution, (b) 4,000k PEO polymer solution. The plot of resolution length was prepared based on the electrophoresis in Figs 1 and 2.
Conclusions
We performed a systematic research on the separation of RNA in PEG/PEO polymer solution with different molecular weight and concentration, and analyzed the separation performance by resolution length. We found three regimes of the RNA migration by the logarithmic plot of mobility versus RNA length in both PEG and PEO solutions. The polymer concentration affected the migration time, resolution between RNA fragments. In addition, the minimum resolution length for RNA in the optimal electrophoretic conditions, including the sieving polymer molecular weight, concentration, electric field strength and capillary length, was about 6.0 nt. Those perceptions for PEG/PEO sieving polymers motivated CE as an effective tool for RNA separations.
Author Contributions
Conceived and designed the experiments: YY ZL. Performed the experiments: CL. Analyzed the data: CL YY DZ. Contributed reagents/materials/analysis tools: ZL XD XZ. Wrote the paper: CL ZL YY.
References
- 1.
Mitchelson KR, Cheng J (2004) Capillary Electrophoresis of Nucleic Acids: Introduction to the capillary electrophoresis of nucleic acids: Springer.
- 2. Guttman A. High-resolution carbohydrate profiling by capillary gel electrophoresis. Nature. 1996; 380: 461–462. pmid:8602248
- 3. Karger BL. High-performance capillary electrophoresis. Nature. 1989; 339: 641–642. pmid:2733797
- 4. Yu Z, Kabashima T, Tang C, Shibata T, Kitazato K, et al. Selective and facile assay of human immunodeficiency virus protease activity by a novel fluorogenic reaction. Anal Biochem. 2010; 397: 197–201. pmid:19852926
- 5. Lines JA, Yu Z, Dedkova LM, Chen S. Design and expression of a short peptide as an HIV detection probe. Biochem Bioph Res Co. 2014; 443: 308–312.
- 6. Yu Z, Schmaltz RM, Bozeman TC, Paul R, Rishel MJ, et al. Selective Tumor Cell Targeting by the Disaccharide Moiety of Bleomycin. J Am Chem Soc. 2013; 135: 2883–2886. pmid:23379863
- 7. Li Z, Li D, Zhang D, Dou X, Yamaguchi Y. Determination and quantification of Escherichia coli by capillary electrophoresis. Analyst. 2014; 139: 6113–6117. pmid:25307062
- 8. Sumitomo K, Mayumi K, Yokoyama H, Sakai Y, Minamikawa H, et al. Dynamic light-scattering measurement of sieving polymer solutions for protein separation on SDS CE. Electrophoresis. 2009; 30: 3607–3612. pmid:19768704
- 9. Sumitomo K, Sasaki M, Yamaguchi Y. Acetic acid denaturing for RNA capillary polymer electrophoresis. Electrophoresis. 2009; 30: 1538–1543. pmid:19340827
- 10. Li Z, Dou X, Ni Y, Chen Q, Cheng S, et al. Is pulsed electric field still effective for RNA separation in capillary electrophoresis? J Chromatogr A. 2012; 122: 274–279.
- 11. Li Z, Dou X, Ni Y, Sumitomo K, Yamaguchi Y. The influence of polymer concentration, applied voltage,modulation depth and pulse frequency on DNA separation by pulsed field capillary electrophoresis. J Sep Sci. 2010; 33: 2811–2817. pmid:20715140
- 12. Yamaguchi Y, Todorov TI, Morris MD, Larson RG. Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions. Electrophoresis. 2004; 25: 999–1006. pmid:15095440
- 13. Li Q, Pei J, Song P, Kennedy RT. Fraction Collection from Capillary Liquid Chromatography and Off-line Electrospray Ionization Mass Spectrometry Using Oil Segmented Flow. Anal Chem. 2010; 84: 5260–5267.
- 14. Anderson GJ, Cipolla C, Kennedy RT. Western Blotting Using Capillary Electrophoresis. Anal Chem. 2011; 83: 1350–1355. pmid:21265514
- 15. Liu P, Seo TS, Beyor N, Shin KJ, Scherer JR, Mathies RA. Integrated Portable Polymerase Chain Reaction-Capillary Electrophoresis Microsystem for Rapid Forensic Short Tandem Repeat Typing. Anal Chem. 2007; 79: 1881–1889. pmid:17269794
- 16. Masubuchi Y, Oana H, Matsumoto M, Doi M, Yoshikawa K. Conformational dynamics of DNA during biased sinusoidal field gel electrophoresis. Electrophoresis. 1996; 17: 1065–1074. pmid:8832173
- 17. Oana H, Masubuchi Y, Matsumoto M, Doi M, Matsuzawa Y, et al. Periodic motion of large DNA molecules during steady field gel electrophoresis. Macromolecules. 1994; 27: 6061–6067.
- 18. Oana H, Masubuchi Y, Matsumoto M, Doi M, Matsuzawa Y, Yoshikawa K. Periodic motion of large DNA molecules during steady field gel electrophoresis. Macromolecules. 1994; 27: 6061–6067.
- 19. Shi X, Hammond RW, Morris MD. Dynamics of DNA during pilsed field electrophoresis in entangled and dilute polymer solutions. Anal Chem. 1995; 67: 3219–3222. pmid:8686887
- 20. Slater GW. Theory of band broadening for DNA gel electrophoresis and sequencing. Electrophoresis. 1993; 14: 1–7. pmid:8462504
- 21. Slater GW, Desruisseaux C, Hubert SJ. DNA separation mechanisms during electrophoresis. METHODS IN MOLECULAR BIOLOGY-CLIFTON THEN TOTOWA-. 2001; 162: 27–42.
- 22. Slater GW, Desruisseaux C, Hubert SJ, Mercier J-F, Labrie J, et al. Theory of DNA electrophoresis: A look at some current challenges. Electrophoresis. 2000; 21: 3873–3887. pmid:11192112
- 23. Todorov TI, Morris MD. Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions. Electrophoresis. 2002; 23: 1033–1044. pmid:11981850
- 24. Todorov TI, Yamaguchi Y, Morris MD. Effect of urea on the polymer buffer solutions used for the electrophoretic separations of nucleic acids. Anal Chem. 2003; 75: 1837–1843. pmid:12713041
- 25. Viovy J, Duke T. DNA electrophoresis in polymer solutions: Ogston sieving, reptation and constraint release. Electrophoresis. 1993; 14: 322–329. pmid:8500463
- 26. Bernard Tinland AP, Jean Sturm, Gilbert Weill. Persistence Length of Single-Stranded DNA. Macromolecules. 1997; 30: 5763–5765.
- 27. Tinoco I Jr, Bustamante C. The effect of force on thermodynamics and kinetics of single molecule reactions. Biophys Chem. 2002; 101: 513–533. pmid:12488024
- 28. Cheng SZD, Chen J. Nonintegral and integral folding crystal-growth in low molecular mass poly(ethylene oxide) fractions. J Polym Sci B Polym Phys. 1991; 29: 311–327.
- 29. Balijepalli S, Schultz J, Lin J. Phase behavior and morphology of poly (ethylene oxide) blends. Macromolecules. 1996; 29: 6601–6611.
- 30. Merdas A, Gindre M, Ober R, Nicot C, Urbach W, Waks M. Nonionic surfactant reverse of C12E4 in dodecane: temperature dependence of size and shape. J Phys Chem. 1996; 100: 15180–15186.
- 31. Briscoe B, Luck P, Zhu S. Rheological study of poly(ethylene oxide) in aqueous salt solutions at high temperature and presure. Macromolecules. 1996; 29: 6208–6211.
- 32. Bieze TWvdM, JRC, Eisenbach CD, Leyte JC. Polymer dynamics in aquarious poly(ethylene oxide) solutions an NMR study. Macromolecules. 1994; 27: 1355–1366.
- 33. Chen J, Cheng SZD, Wu SS, Lotz B, Wittmann J-C. Polymer decoration study in chain folding behaivior of solution-grown poly(ethlene oxide) crystals. J Polym Sci Pol Phys. 1995; 33: 1851–1855.
- 34. Kim Y, Yeung ES. Separation of DNA sequencing fragments up to 1000 bases by using poly (ethylene oxide)-filled capillary electrophoresis. J Chromatogr A. 1997; 781: 315–325. pmid:9368394
- 35. Kim Y, Yeung ES. DNA sequencing with pulsed-field capillary electrophoresis in poly (ethylene oxide) matrix. Electrophoresis. 1997; 18: 2901–2908. pmid:9504828
- 36. Li Z, Dou X, Ni Y, Yamaguchi Y. Separation of long DNA fragments by inversion field capillary electrophoresis. Anal Bioanal Chem. 2011; 401: 1665–1671.
- 37. Hjertén S. High-performance electrophoresis: elimination of electroendosmosis and solute adsorption. J Chromatogr A. 1985; 347: 191–198.
- 38. Li Z, Liu C, Yamaguchi Y, Ni Y, You Q, et al. Capillary electrophoresis of a wide range of DNA fragments in a mixed solution of hydroxyethyl cellulose. Anal Methods-UK. 2014; 6: 2473–2477.
- 39. Li Z, Dou X, Ni Y, Sumitomo K, Yamaguchi Y. Acetic acid denaturing pulsed field capillary electrophoresis for RNA separation. Electrophoresis. 2010; 31: 3531–3536. pmid:20931616
- 40. Li Z, Liu C, Dou X, Ni Y, Wang J, et al. Electromigration behavior of nucleic acids in capillary electrophoresis under pulsed-field conditions. J Chromatogr A. 2014; 1331: 100–107. pmid:24472841
- 41. Barron A, Soane D, Blanch H. Capillary electrophoresis of DNA in uncross-linked polymer solutions. J Chromatogr A. 1993; 652: 3–16. pmid:8281261
- 42. Kan CW, Barron AE. A DNA sieving matrix with thermally tunable mesh size. electrophoresis. 2003; 24: 55–62. pmid:12652572
- 43. Heller C. Principles of DNA separation with capillary electrophoresis. Electrophoresis. 2001; 22: 629–643. pmid:11296917