In-Situ Sampling and Separation of RNA from Individual Mammalian

Chemical Analysis of Single Cells. Laura M. Borland , Sumith Kottegoda , K. Scott Phillips , Nancy L. Allbritton. Annual Review of Analytical Chemistr...
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Anal. Chem. 2000, 72, 4073-4079

In-Situ Sampling and Separation of RNA from Individual Mammalian Cells Futian Han and Sheri J. Lillard*

Department of Chemistry, University of California, Riverside, California 92521

In this investigation RNA was directly sampled and separated at the single-cell level (without extraction) by capillary electrophoresis (CE). Laser-induced fluorescence (LIF) was employed to detect ethidium bromidelabeled RNA molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution included poly(vinylpyrrolidone) to eliminate the electroosmotic flow and mannitol to enhance the separation. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cells were injected into a capillary and the cells were lysed online with sodium dodecyl sulfate (SDS) solutions before running electrophoresis. Low molecular mass (LMM) RNAs as well as larger fragments (tentatively identified as 18S and 28S ribosomal RNA by comparison with the literature) were detected with this system, which corresponds to a detected amount of ≈10-20 pg of RNA/cell. A Proteinase K study showed that proteins incorporated with RNA molecules were eliminated by SDS treatment and thus did not influence the migration of RNA. Experiments were also performed with this technique to detect nucleic acid damage. Changes in the peak pattern were detected in the cells treated with hydrogen peroxide, which meant that strand breaks occurred in DNA and RNA. It was found that 60 mM caused the most severe damage to the nucleic acids. The Human Genome Project has sparked a surge of methods designed for sequencing DNA.1 Because of this, the field of genomics has advanced rapidly in recent years in the analytical community. Another related and emerging field is that of proteomics in which the chemistry and biology of expressed proteins are emphasized.2 However, despite the significant advances in the measurement of DNA (genomics) and the measurement of expressed protein (proteomics),3,4 innovative studies on the intermediate processes involving direct separation and detection of RNA are lacking. Limited applications dealing with detection * Corresponding author. Phone: 909-787-3392. Fax: 909-787-4713. E-mail: [email protected]. (1) Yager, T. D.; Nickerson, D. A.; Hood, L. E. Trends Biochem. Sci. 1991, 16, 454-461. (2) Walsh, B. J.; Molloy, M. P.; Williams, K. L. Electrophoresis 1998, 19, 18831890. (3) Hancock, W.; Apffel, A.; Chakel, J.; Hahnenberger, K.; Choudhary, G.; Traina, J. A.; Pungor, E. Anal. Chem. 1999, 71, 742A-748A. (4) Yates, J. R. Trends Genet. 2000, 16, 5-8. 10.1021/ac000428g CCC: $19.00 Published on Web 07/22/2000

© 2000 American Chemical Society

of mRNA from cell lysates have been reported in which the reverse-transcriptase polymerase chain reaction (RT-PCR) technique was employed.5 However, in RT-PCR, DNA molecules that have been synthesized enzymatically from the mRNA template, and not RNA molecules, are detected. Traditionally, slab gel electrophoresis has been the dominant technique in the characterization of RNA, including Northern Blotting analysis, RT-PCR analysis, and RNA mass determination.6,7 More recently, capillary electrophoresis (CE) has found abundant applications in the separation of biomolecules such as oligonucleotides, proteins, and DNA fragments.8,9 DNA fragments with different numbers of base pairs have identical charge/mass ratios, which necessitates the use of molecular sieving as a separation mode. The mobilities of smaller fragments in the sieving matrix are higher than those of the larger fragments. In many cases, capillaries filled with various crosslinked gels are used to separate DNA fragments.10 However, gelfilled capillaries with good separation efficiencies are very difficult to prepare due to bubble formation, and the storage of these capillaries can be problematic as well. Other drawbacks with these capillaries include poor reproducibility and short lifetimes.11 Nongel sieving polymers are good alternatives for gel capillaries in which entangled polymer solutions are used instead of cross-linked gels. The fluidity of entangled polymer solutions makes it possible to renew the capillary after each run, which results in good reproducibility and long capillary lifetime.12-14 Several kinds of hydrophilic polymers, such as poly(ethylene oxide),15 poly(ethylene glycol),16 poly(vinyl alcohol),17 glucomannan,18 linear (5) See for example (and references therein), Personett, D. A.; Chouinard, M.; Sugaya, K.; McKinney, M. J. Neurosci. Met. 1996, 65, 77-91 or Borson, N. D.; Strausbauch, M. A.; Wettstein, P. J.; Oda, R. P.; Johnston, S. L.; Landers, J. P. BioTechniques 1998, 25, 130-137. (6) Kameda, Y.; Miura, M.; Ohno, S. Brain Res. 2000, 852, 453-462. (7) Melton, D. A.; Krieg, P. A.; Rebagliati, M. R.; Maniatis, T.; Zinn, K.; Green, M. R. Nucleic Acids Res. 1984, 12, 7035-7056. (8) Tran, N. T.; Taverna, M.; Chevalier, M.; Ferrier, D. J. Chromatogr. A 2000, 866, 121-135. (9) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361. (10) Tomita, M.; Okuyama, T.; Hidaka, K.; Ameno, S.; Ameno, K.; Ijiri, I. J. Chromatogr. B 1996, 685, 185-190. (11) Swerdlow, H.; Dew-Jager, K. E.; Brady, K.; Grey, R.; Dovichi, N. J.; Gesteland, R. Electrophoresis 1992, 13, 475-483. (12) Dolnik, V.; Gurske, W. A. Electrophoresis 1999, 20, 3373-3380. (13) Barron, A. E.; Sunada, W. M.; Blanch, H. W. Electrophoresis 1995, 16, 6474. (14) Schell, J.; Wulfert, M.; Riesner, D. Electrophoresis 1999, 20, 2864-2869. (15) Zhang, N.; Yeung, E. S. J. Chromatogr. A 1997, 768, 135-141. (16) Auriola, S.; Ja¨a¨skela¨inen, I.; Regina, M.; Urtti, A. Anal. Chem. 1996, 68, 3907-3911.

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polyacrylamide,19 and cellulose and its derivatives,12,20 have been used as sieving media for the separation of DNA or oligonucleotides. Although DNA separations by capillary electrophoresis have been investigated extensively, only a limited number of applications on RNA determinations have been reported. Using CE with entangled polymer solutions, Katsivela and Hofle explored the separation of low molecular mass (LMM) RNA (including various tRNA and 5S rRNA) extracted from a population of cells. They applied this technique to LMM RNA fingerprinting to identify bacteria.21,22 The separation of larger RNA was achieved under denaturing conditions by Skeidsvoll and Ueland.23 This technique was used for the measurement of total RNA after RNA extraction from human cells. Another study was done using a microscale chip in which Ogura et al. demonstrated the separation of two major components of extracted ribosomal RNA (rRNA) in hydroxypropylmethylcellulose (HPMC) solution with ethidium bromide laser-induced fluorescence (LIF) detection.24 The authors indicated that this method could be used as a quality assessment method for extracted RNA before additional sample preparation steps, such as RT-PCR, were taken. Work by Kolesar et al. showed the direct quantitation of HIV-1 RNA in which CE-LIF was used to detect viral RNA after hybridization with a fluorescently labeled DNA probe.25 More recently, Saevels and co-workers measured the catalytic activity of a hammerhead ribozyme (i.e., a catalytic RNA molecule that functions as an enzyme) by capillary electrophoresis coupled with UV detection.26 It is noteworthy that all of these applications involving RNA determinations were based on samples that were either synthesized, such as the hammerhead ribozyme, or extracted from the cells of interest. When detection sensitivity dictates that RNA from a population of cells is necessary to obtain an adequate amount of sample, then RNA extraction becomes a requirement. If high RNA purity in the absence of contaminating cellular species is required for an experiment, then an extraction step is also necessary.27 However, for many situations, a method in which RNA is directly sampled from one cell at a time would be preferable. When such a method is devised, the complications of additional sample manipulation steps (e.g., extraction) on the integrity of RNA are avoided (i.e., the RNA can remain under native conditions). Another distinct advantage of in-situ RNA sampling from single cells is that the chemical information from one cell is not averaged with the other cells in the population. Such an advantage becomes significant in the area of monitoring populations of cells (17) Kleemiss, M. H.; Gilges, M.; Schomburg, G. Electrophoresis 1993, 14, 515522. (18) Izumi, T.; Yamaguchi, M.; Yonede, K.; Isobe, T.; Okuyama, T.; Shinoda, T. J. Chromatogr. 1993, 652, 41-46. (19) Ruiz-Martinez, M. C.; Salas-Solano, O.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1516-1527. (20) Chen, Y.-H.; Wang, W.-C.; Young, K.-C.; Chang, T.-T.; Chen, S. H. Clin. Chem. 1999, 45, 1938-1943. (21) Katsivela, E.; Hofle, M. G. J. Chromatogr. A 1995, 700, 125-136. (22) Katsivela, E.; Hofle, M. G. J. Chromatogr. A 1995, 717, 91-103. (23) Skeidsvoll J.; Ueland, P. M. Electrophoresis 1996, 17, 1512-1517. (24) Ogura, M.; Agata, Y.; Watanabe, K.; McCormick, R. M.; Hamaguchi, Y.; Aso, Y.; Mitsuhashi, M. Clin. Chem. 1998, 44, 2249-2255. (25) Kolesar, J. M.; Allen, P. G.; Doran, C. M. J. Chromatogr. B 1997, 697, 189194. (26) Saevels, J.; Van Schepdael, A.; Hoogmartens, J. Anal. Biochem. 1999, 266, 93-101. (27) Boedtker, H. Met. Enzymol. 1968, 12B, 429-458.

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for chemical damage or abnormalities. Only a technique capable of single-cell measurements can obtain such information. Many studies of single cells by CE and other methods have been performed; representative references to such articles are cited here.28-34 However, our study is to our knowledge the first time in which RNA has been separated directly from an individual cell. EXPERIMENTAL SECTION Chemicals and Reagents. The hydroxypropylmethylcellulose with low molecular weight (HPMC-5, the viscosity of which is 5 cp in 2% aqueous solution and the molecular weight is ≈10 000) was from Aldrich (Milwaukee, WI). Poly(vinylpyrrolidone), molecular weight 1 000 000, was from Polysciences Inc. (Warrington, PA). Tris(hydroxymethyl)aminomethane, mannitol, and sodium dodecyl sulfate (SDS) were obtained from Sigma (St. Louis, MO). Ethylenediamine tetraacetic acid disodium salt (EDTA), 0.25% trypsin-EDTA, the ΦX 174/Hae III DNA standard, and the RNA ladder were purchased from Life Technologies Inc. (Gaithersburg, MD). The DNA standard contains 11 double-stranded fragments with 72, 118, 194, 234, 271, 281, 310, 603, 872, 1078, and 1353 bp. The DNA sample was diluted with deionized water to a concentration of about 0.04 µg/mL and stored at -20 °C. Samples were diluted to appropriate concentrations with deionized water immediately before use. The RNA ladder consists of fragments of 0.155, 0.280, 0.400, 0.550, 0.780, 1.280, 1.520, and 1.770 kb. The intercalating dye, ethidium bromide, was from Molecular Probes (Eugene, OR) and the enzymes (RNase I and Proteinase K) were from Promega (Madison, WI). All other chemicals, unless otherwise noted above, were from Fisher Scientific (Fairlawn, NJ). Deionized water (resistance g18 MΩ) was prepared with a Milli-Q System (Bedford, MA). Cells and Growth Conditions. The Chinese Hamster Ovary (CHO-K1) cell line was used in our study as a model system to study nucleic acids.35 The cell line was obtained from ATCC (Manassas, VA). The CHO-K1 cells were cultured in an F-12 medium (ATCC) with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 10% fetal bovine serum as supplements. The cells were incubated at 37 °C in an atmosphere of 5% CO2. Cell Preparation. Cells were isolated from a culture flask under sterile conditions 2-3 days after passage. The medium was removed from the flask and the cells were resuspended in 5 mL of 0.25% trypsin-EDTA and incubated for 5 min at 37 °C. The cells were centrifuged at 126g for 5 min, the trypsin-EDTA was aspirated, and the cell pellet was resuspended in phosphatebuffered saline (PBS, containing 9 g/L sodium chloride, 5 mM D-glucose, 1 mM sodium phosphate monobasic, and 3 mM sodium phosphate dibasic; pH was adjusted to 7.4 with 2 M sodium hydroxide). The cells were washed using this PBS procedure a (28) Krylov, S. N.; Starke, D. A.; Arriaga, E. A.; Zhang, Z.; Chan, N. W. C.; Palcic, M. M.; Dovichi, N. J. Anal. Chem. 2000, 72, 872-877. (29) Jung, S.-K.; Gorski, W.; Aspinwall, C. A.; Kauri, L. M.; Kennedy, R. T. Anal. Chem. 1999, 71, 3642-3649. (30) Swanek, F. D.; Chen, G.; Ewing, A. G. Anal. Chem. 1996, 68, 3912-3916, (31) Li, L.; Garden, R. W.; Romanova, E. V.; Sweedler, J. V. Anal. Chem. 1999, 71, 5451-5458. (32) Lillard, S. J.; Yeung, E. S. J. Chromatogr. B 1996, 687, 363-369. (33) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1998, 70, 1164-1170. (34) Hsieh, S.; Jorgenson, J. W. Anal. Chem. 1997, 69, 3907-3914. (35) Iliakis, G. E.; Pantelias, G. E.; Okayasu, R.; Blakely, W. F. Radiation Res. 1992, 131, 192-203.

total of four times to eliminate possible leftover components from the medium and were suspended in PBS buffer again. A 50-µL aliquot of the cell suspension was mixed with Trypan Blue (Life Technologies, 0.02% final Trypan Blue in PBS buffer), and viable cells were counted with a Brightline hemacytometer (Hausser Scientific, Horsham, PA). Approximately 90-95% of these cells treated by Trypan Blue exclusion were viable. Buffer Preparation. The background electrolyte (BGE) for CE was 0.1 M Tris-0.1 M boric acid-2 mM EDTA, pH 8.3 (TBE). The sieving solutions were prepared by adding the polymers to the BGE and stirring the mixture with a magnetic stirrer until all the polymer was dissolved and the solution became homogeneous (≈60 min). This solution does not need to be filtered or degassed before using. Ethidium bromide (0.5 µg/mL) was added to the sieving buffer as an intercalating dye and 6% mannitol was used as an additive to enhance the separation. As shown previously, mannitol appears to interact with HPMC molecules in TBE buffer by forming tetraborate bridges and influencing the dynamic sieving network of the polymer solution.36 In most single-cell experiments 0.2% SDS in the TBE buffer was employed to lyse the cells. However, 0.03% SDS in deionized water was used in the RNase I comparison because 0.2% SDS will deactivate the enzyme (Manufacturer’s Protocol, Promega). A SDS solution with a concentration of 0.1% in deionized water was used in the Proteinase K study because Proteinase K will keep its activity in this solution (Manufacturer’s Protocol, Promega). The lysing buffer for the RNase I study also contained 1× reaction buffer (Promega, diluted from 10× solution containing 100 mM Tris-HCl, pH 7.5; 50 mM EDTA, pH 8.0 and 2 M sodium acetate) and 0.5 U/µL RNase I. Proteinase K was reconstituted to a concentration of 1 mg/mL in a buffer containing 50 mM TrisHCl and 10 mM CaCl2 (pH 8.0). The lysing buffer for the Proteinase K study was prepared by diluting the Proteinase K solution with the 0.1% SDS solution to a concentration of 100 µg/ mL. CE System. A home-built CE-LIF system was used in this study. The high-voltage power supply was from Glassman High Voltage Inc. (Whitehouse Station, NJ). 50-µm i.d. × 40-cm bare fused silica capillaries (360-µm o.d., Polymicro Technologies, Phoenix, AZ) were used for the separation. The polyimide coating was removed by hot sulfuric acid to form the detection window 15 cm from the anodic end of the capillary. The coating at the inlet end of the capillary was also removed for a clear view of cell injection. The anodic high-voltage end of the capillary, to which a voltage of 6 kV was applied to drive the electrophoresis, was isolated in a Plexiglas box as a safety precaution. The injection took place at the cathodic end, which was held at ground potential. DNA standards were injected for 5 s at 6 kV; individual cells were injected as described in the next section. A helium-neon laser (JDS Uniphase Inc., Manteca, CA) was used for excitation at 543.5 nm; the power of the laser is 4 mW. The laser was focused onto the capillary with a 1-cm focal length lens, and the fluorescence was collected with a 10× microscope objective (Edmund Scientific, Barrington, NJ) at a 90° angle to the incident light. The fluorescent image was focused onto a photomultiplier tube (PMT) (R928, Hamamatsu, Bridgewater, NJ). Two long-pass filters (RG610 and OG570, Schott Scientific Glass, (36) Han, F.; Xue, J.; Lin, B. Talanta 1998, 46, 735-742.

Inc., Parkersburg, WV) were mounted to the front of the PMT and used to prevent stray laser light from causing a response. In addition, a spatial filter was placed on top of the optical filter to reject stray light so that only the fluorescence passing through the microscope objective was transmitted. Data were collected by a DT 2804 board, and the electropherogram was generated by ChromPerfect software (Justice Innovations, Inc., Mountain View, CA). The original data files were converted to ASCII, and the electropherograms were replotted using Excel. Cell Injection and Lysing. Cell injection was performed using a Micromaster III microscope (Fisher Scientific) under a 10× objective (100× total magnification). A special mount was constructed to hold the inlet end of the capillary on the microscope stage under the objective. A syringe filled with the sieving buffer was connected to the outlet end of the capillary by plastic tubing and used to fill the capillary and to renew the sieving buffer after each run. Ten microliters of cell suspension was placed on a microscope slide in which the capillary inlet was positioned. For injection, the syringe was gently withdrawn to apply suction and a cell was drawn into the capillary. The microscope slide was moved to another spot while the capillary remained fixed. Ten microliters of the lysing buffer was added to the slide, and lysing buffer was brought slowly into the capillary while the cell was observed under the microscope. In the 0.2% and 0.1% SDS solutions, the cell is lysed within seconds, and in the 0.03% SDS solution, it takes 1-2 min. After the cell was lysed, the two ends of the capillary were placed into the buffer reservoirs and electrophoresis was initiated. In the enzyme studies a 5-20-min reaction time (5 min for the RNase I and 20 min for the Proteinase K) was allowed between lysing and electrophoresis to ensure completion of the enzymatic reaction. RESULTS AND DISCUSSION Investigation of the Sieving Ability. Entangled polymer solutions used in many cases have high viscosity, which can make filling the capillary difficult.37 Han et al. have developed a sieving matrix based on a low molecular weight HPMC to separate DNA fragments.38 This sieving matrix has very low viscosity and high separation efficiency, but a coated capillary must be used to eliminate the influence of electroosmotic flow. Yeung’s group has found that poly(vinylpyrrolidone), PVP, has a very low viscosity as a sieving medium for DNA separation and sequencing.39 PVP also has an excellent self-coating property, which means it can form a dynamic coating on the wall of a bare capillary and suppress the electroosmotic flow. Although the separation efficiency of PVP was not adequate in our study (data not shown), the wall-coating properties of PVP were desirable. Thus, we combined PVP with HPMC-5 and developed a matrix that can provide high sieving efficiency without precoating the capillary. The sieving buffer consists of 1% HPMC-5, 0.5% PVP, 6% mannitol, and 0.5 µg/mL ethidium bromide. To investigate the separation efficiency of the sieving matrix, we used ΦX 174/Hae III as a DNA standard. Figure 1 shows the separation of the DNA standard in the dualpolymer sieving system. All 11 fragments were well-resolved within 25 min and the sensitivity and reproducibility of this system are (37) Chiari, M.; Nesi, M.; Fazio, M.; Righetti, P. G. Electrophoresis 1992, 13, 690-697. (38) Han, F.; Huynh, B.; Ma, Y.; Lin, B. Anal. Chem. 1999, 71, 2385-2389. (39) Gao, Q.; Yeung, E. S. Anal. Chem. 1998, 70, 1382-1388.

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Figure 1. Electropherogram of ΦX 174/Hae III DNA fragments. Sieving buffer: 1% HPMC-5, 0.5% PVP, 6% mannitol, and 0.5 µg/ mL ethidium bromide in TBE (0.1 M Tris-0.1 M boric acid, 2 mM EDTA, pH 8.3) buffer. Capillary: 40 cm × 50 µm i.d. (360-µm o.d.) with 25-cm effective length. Injection: electrokinetic injection at -6 kV for 5 s. Electrophoresis voltage: -6 kV. LIF detection: He-Ne laser excitation at 543.5 nm. Other conditions as listed in the Experimental Section.

Figure 2. (A) Electropherogram of RNA from a single CHO-K1 cell (control). Cell lysis was achieved with 0.03% SDS and 1× reaction buffer. (B) Electropherogram of a single CHO-K1 cell after RNase I treatment. The same lysing buffer was used except that this lysing buffer also contained 0.5 U/µL RNase I. A 5-min incubation time was allowed for both studies after cell lysing (before electrophoresis) to ensure complete enzymatic reaction and to ensure direct comparison for the control study. Other conditions were the same as those in Figure 1.

excellent. The largest fragment (1353 bp) of a 2 ng/mL sample gave a peak with a signal/noise ratio of over 350. This DNA standard (2 ng/mL) represents a sample 500-fold less concentrated than that used by Ogura et al. in which 1 ng/µL of the same DNA marker gave peaks that were barely discernible.24 Thus, our results showed much better sensitivity regarding concentration detection limit, by nearly 4 orders of magnitude, compared to the microchip system.24 We found that the RSD of the migration time of this fragment in seven consecutive injections is 0.28%. We also did not notice any dose-related shifts of the migration time (i.e., changes in migration time as a function of DNA concentration), which indicates that the reproducibility of migration times in our system is better than the microchip format.24 Single-Cell RNA Sampling and Separation. As mentioned previously, most of the RNA studies with CE have used extracted RNA as the sample and UV absorbance to detect the separated fragments. RNA extraction is very time-consuming, labor-intensive, and expensive. UV absorbance is a detection technique with relatively low sensitivity. To detect RNA at the single-cell level, laser-induced fluorescence was employed in our study. It is widely known that ethidium bromide is an intercalating dye, which can bind into G-C base pairs and form a DNA-dye complex with very high fluorescence efficiency.40 Although RNA is not a double helix like DNA is, parts of the single-stranded RNA molecule may fold and form intramolecular base-paired regions, giving rise to complex secondary structure.41 This property makes it possible to label RNA with ethidium bromide. The separation of RNA directly sampled from a single cell (without extraction) is shown in Figure 2 (trace A). Five peaks were obtained within 25 min. Those peaks were confirmed to be from RNA by an additional RNase I experiment. RNase I is an enzyme which degrades RNA into its constituent bases.42 Because ethidium bromide is selective

for base-paired nucleic acids (e.g., DNA or RNA), we already know that the peaks in Figure 2A must be DNA or RNA. The addition of RNase I will cause degradation and disappearance of RNA peaks and will leave peaks from DNA unaltered. The introduction of RNase I was performed by adding the enzyme into the lysing buffer. After cell lysis, the RNase became accessible to the intracellular RNA. Electrophoresis was allowed to proceed and every peak disappeared after RNase I treatment as shown in trace B of Figure 2. Because a whole cell was introduced into the capillary, without extraction, all fractions of RNA should be present. However, we expect that peaks corresponding to mRNA (≈3% of total RNA) are not detected because mRNA molecules are very unstable and most likely are degraded under our conditions.43 Some of the rRNAs (5S and 5.8S fractions) and tRNA migrate close to each other because they have similar molecular weights (hence, it is believed that the early group of peaks contains these RNA fractions). The two largest fragments elute last and are 18S rRNA and 28S rRNA, respectively, as compared to those in the literature.23,24 Furthermore, the expected amount of total RNA in a single cell can be estimated from the literature. From studies utilizing isolated total RNA, it is estimated that 1 µg results from (5 × 104)-(1 × 105) mammalian cells, which corresponds to 1020 pg of RNA/cell.24, 44 It is interesting to note that the RNase experiment shows there are no peaks corresponding to DNA in the control cell runs. Our analysis conditions are gentle and include low detergent concentrations for lysis and the absence of shear forces resulting from pipetting or centrifuging solutions during a sample preparation or isolation step. Because of the mild environment, genomic DNA is not degraded physically into smaller fragments and probably

(40) Waring, M. J. J. Mol. Biol. 1965, 13, 269-282. (41) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Company: New York, 1988; pp 92-98. (42) Meador, J.; Cannon, B.; Cannistraro, V. J.; Kennell, D. Eur. J. Biochem. 1990, 187, 549-553.

(43) Davidson, V. L.; Sittman, D. B. Biochemistry, 3rd ed.; Harwal Publishing: Philadelphia, 1994; pp 179-181. (44) Kawasaki, E. S.; Wang, A. M. In PCR Technology: Principles and Applications for DNA Amplification; Erlich, H. A., Ed.; Stockton Press: New York, 1989; p 92.

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Figure 3. Electropherogram of an RNA ladder. Other conditions were the same as those in Figure 1.

exists as megabase- (Mb-) sized molecules, which have considerably low mobilities. To increase speed and resolution of Mb-sized DNA separation, pulsed-field slab gel electrophoresis is used by most researchers.45 Although we extended the running time to 120 min to permit enough time for DNA migration, no extra peaks were observed within this 2-h period (data not shown). The reproducibility of single-cell injection is quite good, considering there is some variability in the position of the cell in the capillary inlet. Six injections in two capillaries showed a 0.24% RSD in the variation of the migration time of the last peak, which was identified to be 28S rRNA compared to the literature.23,24 The fourth peak was tentatively identified to be 18S rRNA by comparison to the same literature.23,24 It is already known that conformation or higher level structure will influence the migration of DNA fragments in some cases, which makes it difficult to determine the fragment length by migration time only.46 A denaturing environment should always be used to determine the fragment length of DNA by a calibration method (i.e., comparing to migration times of markers). In Skeidsvoll’s study, extremely denaturing conditions were used to eliminate the influence of the RNA conformation on migration.23 Denaturant is also necessary when estimating RNA mass information because anomalous migration caused by inter- and intramolecular structures in RNA will influence the accuracy of the estimation.47 In our study, denaturing conditions were not used because of the necessity of instantaneous labeling following cell lysis. Ethidium bromide intercalates quickly and its use is a compromise between sensitivity and reaction rate. Figure 3 illustrates the separation of an RNA ladder in our study. The separation profile is clearly more complex than DNA standards and exhibits poorly resolved peaks. This is further demonstration of the influence of secondary structure on RNA migration. Although good separation of an RNA molecular mass marker was obtained by Skeidsvoll with denatured conditions, they also noticed bad separation of the same marker under native (i.e., nondenatured) conditions.23 Katsivela, however, employed conformation differences to differentiate RNA mobilities because (45) Maercker, C.; Kortwig, H.; Lipps, H. J. Genome Res. 1999, 9, 654-661. (46) Han, F.; Xu, Q.; Lin, B.; Shen, Y.; Wu, G. Chromatographia 1999, 49, 179184. (47) Richwood, D.; Hames, B. D. Gel Electrophoresis of Nucleic Acids: A Practical Approach; IRL Press Ltd.: Eynsham, 1982; pp 1-38.

LMM RNA molecules have similar fragment lengths (number of bases), and separation based on molecular size may not be successful.21,22 This is why the LMM RNAs were not resolved very well under Skeidsvoll’s denatured conditions. Native conditions were used in our experiments to separate RNA directly from a single cell in which LMM RNA was better resolved than in the studies under denatured conditions. These results are promising and should permit detailed information or LMM RNA fingerprinting information to be obtained in future studies. In vivo, ribosomal RNA molecules are incorporated with protein molecules in particles called ribosomes.48 Because it was not clear if the levels of detergent in our study are sufficient to detach protein from RNA,49 the influence of protein on the migration of RNA molecules was also investigated. To determine if there are any proteins still incorporated with RNA after SDS treatment, a Proteinase K study was designed. Proteinase K is an enzyme with protease activity, which degrades proteins. Any proteins bound to RNA should be removed upon enzyme treatment and the protein’s contribution to RNA migration can be assessed. A 20min reaction time was allowed after cell lysing before running electrophoresis to ensure sufficient time for the enzymatic reaction to proceed. We did not observe any difference between the runs with and without Proteinase K treatment (data not shown), which indicates that SDS is efficient in removing associated protein and there is no contribution of protein to RNA migration. Nucleic Acid Damage. Techniques that can provide information about DNA damage are very important in environmental sciences and medical sciences to evaluate environmental toxicology, carcinogenesis, and aging.50,51 Some chemical genotoxins, such as vanadium pentoxide,52 N-methyl-N′-nitro-N-nitrosoguanidine,53 sodium ascorbate,54 and hydrogen peroxide,55 can induce DNA breaks. Nonchemical agents such as UV, X-ray, and γ-irradiation can also cause breaks and other damage to DNA.56-58 Detecting the damage of DNA at the single-cell level is very important because some genotoxins are tissue-specific in their ability to produce strand breaks.59-61 Conventionally, a technique called single-cell gel electrophoresis (or comet assay), first developed by Rydberg and Johanson,62 is employed to investigate DNA damage in single cells. The comet assay requires intensive work and takes at least several hours. Although DNA breaks (by (48) Zubay, G. Biochemistry, 3rd ed.; Wm. C. Brown Publishers: Dubuque, 1993; pp 794-799. (49) Noll, H.; Stutz, E. Met. Enzymol. 1968, 12B, 129-155. (50) Ames, B. N. Science 1983, 221, 1256-1264. (51) Cerutti, P. A. Science 1985, 227, 375-381. (52) Rojas, E.; Valverde, M.; Herrera, L. A.; Altamirano-Lozano, M.; OstroskyWegman, P. Mutat. Res. 1996, 359, 77-84. (53) Yendle, J. E.; Tinwell, H.; Elliott, B. M.; Ashby, J. Mutat. Res. 1997, 375, 125-136. (54) Singh, N. P. Mutat. Res. 1997, 376, 195-203. (55) Collins, A. R.; Dobson, V. L.; Duˇsinska´, M.; Kennedy, G.; Sˇ teˇtina, R. Mutat. Res. 1997, 375, 183-193. (56) Bock, C.; Dittmar, H.; Gemeinhardt, H.; Bauer, E.; Greulich, K.-O. Mutat. Res. 1998, 408, 111-120. (57) Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L. Exp. Cell Res. 1988, 175, 184-191. (58) Ostling, O.; Johanson, K. J. Biochem. Biophys. Res. Commun. 1984, 123, 291-298. (59) Ono, T.; Okada, S. Exp. Gerontol. 1976, 11, 127-132. (60) Lewensohn, R.; Ringborg, U.; Baral, E.; Lambert, B. J. Cell Sci. 1982, 54, 69-78. (61) Niedermuller, H.; Hofecker, G.; Skalicky, M. Mech. Ageing Dev. 1985, 29, 221-238.

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Figure 5. Sample dose-response curve of nucleic acid damage of individual CHO-K1 cells vs hydrogen peroxide concentration.

Figure 4. (A) Electropherogram of a single CHO-K1 cell after exposure to 60-mM hydrogen peroxide for 30 min at room temperature. Other conditions were the same as those in Figure 2. (B) Electropherogram showing separation of nucleic acids from an RNase I-treated single CHO-K1 cell following a 5-min incubation with hydrogen peroxide.

hydrogen peroxide and other agents) are well-known and understood, a logical question is if such agents will cause damage to RNA. In this study we have investigated the possibility of using CELIF to measure the damage of nucleic acids, such as DNA and RNA, inside a single cell. Hydrogen peroxide was employed as an agent to induce nucleic acid damage. The cells were incubated with different concentrations of hydrogen peroxide at room temperature for a fixed exposure time of 30 min. An exposed cell was then injected into the capillary, and cell lysing and electrophoresis were carried out immediately after injection. It was found that hydrogen peroxide exposure resulted in some changes in the peak pattern for single-cell nucleic acids as shown in Figure 4A. The relative fluorescence intensity of the smaller fragments (around 16 min) became higher, and the relative fluorescence intensity of the larger fragments (around 23 min) became smaller. There are several phenomena that could give rise to these changes in signal intensities. If hydrogen peroxide is causing strand breaks, then larger nucleic acid fragments, such as RNA, are being broken (62) Rydberg, B.; Johanson, K. J. In DNA Repair Mechanisms; Hanawalt, P. C., Friedberg, E. C., Fox, C. F. , Eds.; Academic Press: New York, 1978; pp 465-468.

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down to smaller fragments. Thus, if there are more molecules in the smaller size range, the intensity will increase. As mentioned previously, we have tentatively identified the last two peaks in Figure 2A as the 18S and 28S rRNA molecules. In addition to strand breaks in the RNA molecules, hydrogen peroxide will also induce breakage in genomic DNA, as reported previously by others.63,64 Therefore, we cannot say with absolute certainty that all of the toxin-induced fragments are only RNA. To investigate if the enhancement in peak intensity of the smaller fragments includes a contribution from fragmented DNA, an RNase I study was performed in the presence of hydrogen peroxide. The cells were first treated with hydrogen peroxide for 5 min and then the injected cell was lysed with a lysing buffer containing RNase I. Because the peaks from RNA will be eliminated after RNase I treatment, any remaining peaks must be due to DNA. As shown in Figure 4B, it was found that there are still some smaller peaks around 16 and 23 min, which are attributed to damaged (i.e., fragmented) DNA. Figure 5 is an example of a dose-response curve of individual CHO-K1 cells under different levels of hydrogen peroxide exposure. The molar concentration of ethidium bromide intercalated into RNA depends on the size of the RNA molecule (i.e., larger polymers intercalate more dye molecules, hence, exhibiting greater fluorescence). Therefore, because peak area does not always reflect the molar concentration of RNA, absolute peak areas were not used to indicate the extent of damage. Instead, the area ratio (AR) is defined as

AR ) Ai/Aj

(1)

where Ai is the sum of the peak areas of smaller fragments (i.e., between 12 and 17 min) and Aj is the sum of the peak areas of the larger fragments (i.e., >20 min). The AR indicates the extent of total nucleic acid breakage. The more breaks that occurred, the higher the AR and vice versa. Our results showed a bellshaped curve for the CHO-K1 cells. The most severe damage occurred when the hydrogen peroxide concentration was around 60 mM as evidenced by the maximum in the dose-response (63) Pool-Zobel, B. L.; Leucht, U. Mutat. Res. 1997, 375, 105-115. (64) Horva´thova´, E.; Slamenova´, D.; Hlincı´kova´, L.; Mandal, T. K.; Ga´belova´, A.; Collins, A. R. Mutat. Res. 1998, 409, 163-171.

curve. The extent of the damage to the nucleic acids increased when the concentration of hydrogen peroxide was raised from 0 to 60 mM, and more interestingly less damage was detected when the hydrogen peroxide concentration was above 60 mM. The reason for this phenomenon is not totally understood. Cell death in a high concentration of hydrogen peroxide was considered,65 but a Trypan Blue exclusion showed that 80 mM hydrogen peroxide was not cytotoxic to this CHO-K1 cell line (within 60 min). Another possibility is that the relative amounts of DNA and RNA breaks are changing independently of one another, which leads to the observed net dose-response curve. Our technique, however, cannot differentiate between the damage of the two types of nucleic acids. This preliminary study of nucleic acid damage at the single-cell level by capillary electrophoresis provided useful information that can be used in further studies. For example, this CE-based technique may be a promising method to study the damage of RNA by ribotoxins, a class of peptides found in various kinds of fungi that will cause damage and/or breaks in RNA molecules.66,67 (65) Gelvan, D.; Moreno, V.; Clopton, D. A.; Chen, Q. Saltman, P. Biochem. Biophys. Res. Commun. 1995, 206, 421-428. (66) Lin, A.; Huang, R.-G. BioTechniques 1994, 17, 636-638. (67) Kao, R.; Davies, J. FEBS Lett. 2000, 466, 87-90.

CONCLUSIONS A high-sensitivity method was developed to measure RNA, in situ (without extraction), at the single-cell level. Preliminary results showed that this technique may be satisfactory to investigate the damage of nucleic acids caused by genotoxins and/or ribotoxins. More work needs to be done to enhance separation of the LMM RNAs, which once achieved, may become a convenient method to identify individual cells by LMM RNA fingerprinting. ACKNOWLEDGMENT This work was supported by CRCC (University of California Cancer Research Coordinating Committee). We acknowledge technical help from Christopher E. McCoy. We sincerely thank Prof. Werner G. Kuhr for lending us the high-voltage power supply and for his helpful suggestions on the manuscript.

Received for review April 14, 2000. Accepted June 19, 2000. AC000428G

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