Direct Quantification of Single-Molecules of MicroRNA by Total

Jul 23, 2010 - Enrico Giampieri , Marco De Cecco , Daniel Remondini , John Sedivy , Gastone Castellani. PLOS ONE 2015 10 (6), e0118442 ...
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Anal. Chem. 2010, 82, 6911–6918

Direct Quantification of Single-Molecules of MicroRNA by Total Internal Reflection Fluorescence Microscopy Ho-Man Chan,† Lai-Sheung Chan,‡ Ricky Ngok-Shun Wong,‡ and Hung-Wing Li*,† Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, P.R. China, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, P.R. China MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRTPCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis. MicroRNAs (miRNAs) are categorized as a class of small noncoding RNAs with approximately 19-23 nucleotides. It serves as the gene expression and cell development regulator in animals, plants, and viruses by interfering protein synthesis.1,2 Evidences indicated that miRNAs play important roles in cell proliferation, differentiation, and apoptosis.2-4 Recent researches have also explored the differential expression levels of miRNAs in cancerous * Corresponding author: (phone) +852-3411-7065; (fax) +852-3411-7348; (e-mail) [email protected]. † Department of Chemistry, Hong Kong Baptist University. ‡ Department of Biology, Hong Kong Baptist University. (1) Ambros, V. Cell 2003, 113, 673–676. (2) Ambros, V. Nature 2004, 431, 350–355. (3) Carthew, R. W. Curr. Opin. Genet. Dev. 2006, 16, 203–208. (4) Nakahara, K.; Carthew, R. W. Curr. Opin. Cell Biol. 2004, 16, 127–133. 10.1021/ac101133x  2010 American Chemical Society Published on Web 07/23/2010

and noncancerous tissues and displayed its roles as tumor suppressors or oncogenes.5-7 Tumor formation caused by miRNAs is notable since it has been proven that a single sequence of miRNA can regulate multiple gene targets. In the meanwhile, a single gene target can also be regulated by multiple miRNAs. Therefore, on account of the connections between miRNA and cancer development, profiling of miRNA expression levels is proposed as a vital tool for the preliminary diagnosis and prognosis of cancer.8 A majority of the miRNA detection methods are based on the approach of hybridization, in which target molecules of interest arecapturedorhybridizedwiththecomplementaryoligonucleotides.8,9 Hereafter, the frequency of hybridization events are presented in form of measurable signals for the quantification of miRNA. However, the detection of the small size and trace amount of miRNAs is always challenging. Northern blotting is the most prevalent and accredited method for the quantification of miRNAs.10 The technique allows multiplex detection, but it is laborious and sample intensive. Another convincing technique for the determination of miRNA is the real-time polymerase chain reaction (qRT-PCR).11-13 With PCR, copies of specific miRNAs and their corresponding signals are amplified and intensified, respectively. Nonetheless, the short length of target miRNA and intricate design of primer limited the reliability of amplification and labeling.8 Other amplification strategies have also been proposed in recent years aiming at higher detection sensitivity, specificity, and lower consumption of starting materials. For instance, nanoparticles such as OsO2,14 quantum dots,15 and gold15,16 are chemically labeled on detecting probes as signal transducers and amplifiers. Besides, (5) Calin, G. A.; Croce, C. M. Nat. Rev. Cancer 2006, 6, 857–866. (6) Esquela-Kerscher, A.; Slack, F. J. Nat. Rev. Cancer 2006, 6, 259–269. (7) Volinia, S.; Calin, G. A.; Liu, C. G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M.; Roldo, C.; Ferracin, M.; Prueitt, R. L.; Yanaihara, N.; Lanza, G.; Scarpa, A.; Vecchione, A.; Negrini, M.; Harris, C. C.; Croce, C. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2257–2261. (8) Wark, A. W.; Lee, H. J.; Corn, R. M. Angew. Chem., Int. Ed. 2008, 47, 644–652. (9) Cissell, K. A.; Deo, S. K. Anal. Bioanal. Chem. 2009, 394, 1109–1116. (10) Lagos-Quintana, M.; Rauhut, R.; Lendeckel, W.; Tuschl, T. Science 2001, 294, 853–858. (11) Chen, C. F.; Ridzon, D. A.; Broomer, A. J.; Zhou, Z. H.; Lee, D. H.; Nguyen, J. T.; Barbisin, M.; Xu, N. L.; Mahuvakar, V. R.; Andersen, M. R.; Lao, K. Q.; Livak, K. J.; Guegler, K. J. Nucleic Acids Res. 2005, 33, 9. (12) Raymond, C. K.; Roberts, B. S.; Garrett-Engele, P.; Lim, L. P.; Johnson, J. M. RNA 2005, 11, 1737–1744. (13) Schmittgen, T. D.; Jiang, J. M.; Liu, Q.; Yang, L. Q. Nucleic Acids Res. 2004, 32, e43. (14) Gao, Z. Q.; Yang, Z. C. Anal. Chem. 2006, 78, 1470–1477.

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enzymatic amplification techniques such as RAKE assay,17 bioluminescence-enzyme labeling,18 and rolling-circle amplification (RCA)19 are also reported for sensitivity enhancement. These reported detection methods bring great benefit and improvement to the sensitivity of miRNA profiling to certain extends. Nevertheless, sample pretreatment and the modifications may result in loss of samples throughout the multiple pretreatment steps such as sample enrichment, labeling, and purification. The detection sensitivity and reliability are hence hindered. Since cellular miRNA concentration can be as low as 1000 molecules per cell,20 deficiencies in sensitivity may result in unsuccessful quantification of low-abundance miRNAs and, thus, false diagnostic result. Therefore, an ultrasensitive miRNA-profiling assay without the need of pretreatment is demanded. Single-molecule detection (SMD) technique has been widely applied in the behavioral study of individual biomolecules.21,22 Scientific issues such as monitoring enzymatic kinetics,23,24 DNA adsorption/desorption behavior,25,26 verification of nucleic acid hybridization,27 DNA mismatch discrimination,28 protein and DNA conformation dynamics,29,30 and DNA mapping31,32 have successfully been accomplished with SMD. Besides, several groups have also demonstrated the competence of SMD in the quantitation of biomolecules such as proteins33 and viral DNA34,35 by singlemolecule counting. Recently, quantitation of miRNA in singlemolecule level was displayed by Neely and co-workers.36 The novel miRNA detection assay is free of sample enrichment and (15) Liang, R. Q.; Li, W.; Li, Y.; Tan, C. Y.; Li, J. X.; Jin, Y. X.; Ruan, K. C. Nucleic Acids Res. 2005, 33. (16) Yang, W. J.; Li, X. B.; Li, Y. Y.; Zhao, L. F.; He, W. L.; Gao, Y. Q.; Wan, Y. J.; Xia, W.; Chen, T.; Zheng, H.; Li, M.; Xu, S. Q. Anal. Biochem. 2008, 376, 183–188. (17) Nelson, P. T.; Baldwin, D. A.; Scearce, L. M.; Oberholtzer, J. C.; Tobias, J. W.; Mourelatos, Z. Nat. Methods 2004, 1, 155–161. (18) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80, 2319–2325. (19) Cheng, Y.; Zhang, X.; Li, Z.; Jiao, X.; Wang, Y.; Zhang, Y. Angew. Chem., Int. Ed. 2009, 48, 3268–3272. (20) Lim, L. P.; Lau, N. C.; Weinstein, E. G.; Abdelhakim, A.; Yekta, S.; Rhoades, M. W.; Burge, C. B.; Bartel, D. P. Genes Dev. 2003, 17, 991–1008. (21) Joo, C.; Balci, H.; Ishitsuka, Y.; Buranachai, C.; Ha, T. Annu. Rev. Biochem. 2008, 77, 51–76. (22) Weiss, S. Science 1999, 283, 1676–1683. (23) Li, H. W.; Yeung, E. S. Anal. Chem. 2005, 77, 4374–4377. (24) Li, J. W.; Yeung, E. S. Anal. Chem. 2008, 80, 8509–8513. (25) Kang, S. H.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2001, 73, 1091– 1099. (26) Li, H. W.; Park, H. Y.; Porter, M. D.; Yeung, E. S. Anal. Chem. 2005, 77, 3256–3260. (27) Kang, S. H.; Kim, Y. J.; Yeung, E. S. Anal. Bioanal. Chem. 2007, 387, 2663–2671. (28) Gunnarsson, A.; Jonsson, P.; Marie, R.; Tegenfeldt, J. O.; Hook, F. Nano Lett. 2008, 8, 183–188. (29) Cohen, A. E.; Moerner, W. E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12622–12627. (30) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555–559. (31) Chan, E. Y.; Goncalves, N. M.; Haeusler, R. A.; Hatch, A. J.; Larson, J. W.; Maletta, A. M.; Yantz, G. R.; Carstea, E. D.; Fuchs, M.; Wong, G. G.; Gullans, S. R.; Gilmanshin, R. Genome Res. 2004, 14, 1137–1146. (32) Xiao, M.; Phong, A.; Ha, C.; Chan, T. F.; Cai, D. M.; Leung, L.; Wan, E.; Kistler, A. L.; DeRisi, J. L.; Selvin, P. R.; Kwok, P. Y. Nucleic Acids Res. 2007, 35, e16. (33) Tessler, L. A.; Reifenberger, J. G.; Mitra, R. D. Anal. Chem. 2009, 81, 7141– 7148. (34) Lee, J. Y.; Li, J. W.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089. (35) Li, J. W.; Lee, J. Y.; Yeung, E. S. Anal. Chem. 2006, 78, 6490–6496. (36) Neely, L. A.; Patel, S.; Garver, J.; Gallo, M.; Hackett, M.; McLaughlin, S.; Nadel, M.; Harris, J.; Gullans, S.; Rooke, J. Nat. Methods 2006, 3, 41–46.

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amplification, but continuous sample flow is needed for improvement in sensitivity. This microfluidic-assisted fluorescence correlation spectroscopy platform is also comparatively sophisticated. In this article, we present a quantitative single-molecule detection of miRNAs using total internal reflection fluorescence microscopy (TIRFM). For the proof of concept, we have chosen hsa-miR-21 (miR-21) as our detection target. The miR-21 is known as one of the most significant miRNAs elevated in at least six types of cancers including breast, colon, lung, pancreas, prostate, and stomach cancers.7 Studies have indicated the miR-21 mediated tumor growth by serving as an oncogene37 and targeting the tumor suppressor genes such as TPM1 and PDCD 4 in invasion and metastasis.38-40 Compared to the conventional methods, the developed assay here is straightforward because no pretreatment steps are involved. Both the probe and target oligonucleotides are free of chemical modifications. The YOYO-1 labeled miRNA hybrids diffuse freely on unmodified coverslips and are monitored by electron-multiplying charge-coupled device (EMCCD) under TIRFM. TIRFM is a highly sensitive microscopic technique that has been used for SMD in solution. The total internal reflection (TIR) generates evanescent field layer that has a penetration depth of about 100-300 nm depending on the incident angle of the excitation laser beam. The excitation of fluorophores is confined within the evanescent field layer such that background signal from the bulk is greatly suppressed. Herein, the diffusing hybrids are observed as single fluorescent spots when they enter the excitation volume and are excited. Image of fluorescent molecules are acquired for single-molecule counting. The counted number is found to be proportional to the quantity of miRNAs in bulk solution. The developed assay was also employed for the determination of miR-21 in normal and cancerous cell lines and the results were validated with that of qRT-PCR detection. EXPERIMENTAL SECTION Slide Pretreatment. All coverslips were prewashed prior to experiments. Briefly, No. 1 22-mm square cover glasses (Gold Seal, Electron Microscopy System, Hatfield, PA) were sequentially sonicated for 30 min in household detergent, 30 min in acetone (AR grade, Labscan), and 30 min in absolute ethanol. The slides were then successively soaked for 30 min in Piranha solution (H2SO4/30% H2O2) (v/v 1:1), rinsed with distilled water extensively, sonicated for 30 min in HCl/30% H2O2/H2O (v/ v/v 1:1:1) solution, sonicated in distilled water for 15 min, further sonicated for 30 min in Piranha solution, and finally sonicated for 15 min in distilled water twice. The slides were stored in distilled water and blow-dried with nitrogen before use. Preparation of Hybridization Buffers. A 1× Tris-NaCl-EDTA (TNE) buffer containing 20 mM pH 8.0 Tris-HCl (Invitrogen, Carlsbad, CA), 1 mM EDTA, and various concentration (0, 50, 150, 250, and 500 mM) of sodium chloride was prepared with DEPC-treated water (Ambion, Austin, TX) accordingly as the (37) Si, M. L.; Zhu, S.; Wu, H.; Lu, Z.; Wu, F.; Mo, Y. Y. Oncogene 2007, 26, 2799–2803. (38) Lu, Z.; Liu, M.; Stribinskis, V.; Klinge, C. M.; Ramos, K. S.; Colburn, N. H.; Li, Y. Oncogene 2008, 27, 4373–4379. (39) Zhu, S. M.; Si, M. L.; Wu, H. L.; Mo, Y. Y. J. Biol. Chem. 2007, 282, 14328– 14336. (40) Zhu, S. M.; Wu, H. L.; Wu, F. T.; Nie, D. T.; Sheng, S. J.; Mo, Y. Y. Cell Res. 2008, 18, 350–359.

dilution and hybridization buffer. The pH of the TNE buffers was adjusted to 7.4 by addition of 1 M HCl dropwisely. The buffer solution was then filtered through a 0.22 µm nylon membrane filter, autoclaved, and photobleached with UV-C lamp overnight prior to use. Preparation of Probe and Target MicroRNA Oligonucleotides. Commercial available LNA-modified oligonucleotide probe (miRCURY LNA microRNA Detection Probe, Product no: 38102-00) (3′-TCAACATCAGTCTGATAAGCTA-5′) specific to hsa-miR-21 was purchased from Exiqon (Denmark). Two HPLC-purified synthetic DNA oligonucleotides having the complementary sequence to hsa-miR-21 (3′-TCAACATCAGTCTGATAAGCTA-5′) and the mature sequence of hsa-miR-214 (3′-ACAGCAGGCACAGACAGGCAGU-5′) were custom-designed and obtained from Invitrogen (Hong Kong) as the DNA probe and the negative control of the experiments. Two Anti-miR miRNA inhibitors of miR-21 (AM17000, Product ID: AM10206, 3′-CAACACCAGUCGAUGGGCUGU-5′), and anti-miR-21 (AM17000, Product ID: AM12979, 3′-UAGCUUAUCAGACUGAUGUUGA-5′), were purchased from Ambion, acting as the RNA probe and target miR-21 strands, respectively. All oligonucleotides were suspended in 1 µL of DEPC-treated water (Ambion) and further diluted into appropriate concentration with 1× TNE buffer. The melting temperatures (Tm) of the oligonucleotides were predicted using the Probe Tm Predictor accessible online (www.exiqon.com) based on the thermodynamic nearest neighbor model. Optimization of Hybridization Conditions. Ionic Strength. For the determination of optimum hybridization ionic strength, sodium chloride concentrations from 0 to 500 mM in the TNE buffer were used throughout the oligonucleotides dilution and hybridization mixture preparation. Selection of Probe. For the selection of optimal probe with the best hybridization affinity toward miRNA, probes of DNA, RNA and LNA were prepared and hybridized with target miR-21 as described below. In addition, two control experiments including probes only and negative control miR-214 were also performed. Hybridization Time. For the determination of optimum hybridization time, hybrids of same concentration and ionic strength (250 mM NaCl) were incubated for 15 min, 30 min, 1 and 3 h respectively. Hybridization and Labeling of MicroRNA. The hybridization and fluorescence labeling of miRNAs were performed according to the following procedures. LNA probe, DNA probe, RNA probe, miRNA target, and DNA negative control oligonucleotides were diluted with 1× TNE buffers (pH 7.4) to 300 pM in concentration, respectively. The hybridization cocktail contained 15 µL of 300 pM probe strand, 15 µL of appropriate concentration of target miR-21 strand, and 14 µL of TNE buffer. The cocktail was incubated in form of free-diffusing solutions in dry bath (AccuBlock Digital Dry Bath D1100, Labnet, NJ) for 1 h. The hybridization temperature was set to be 20 °C below the Tm of the probe, that is, 52, 47, and 36 °C for LNA, DNA, and RNA probe, respectively. After incubation, 1 µL of 100 nM YOYO-1 Iodide (YOYO) (Invitrogen) was added subsequently to label the hybrids. YOYO labels the hybrids in a dye to base pair (dye/bp) ratio of 1:1. The mixture was kept for 5 min in order to achieve equilibrium and 10 µL of solution was pipetted to the precleaned coverslips for TIRF imaging.

External Calibration of MicroRNA. A calibration curve was established to correlate number of detected single fluorescent spots and concentration of miRNAs. Synthetic miRNA with final concentration of 100, 75, 50, 25, 10, 5, and 1 pM target miR-21 strand was hybridized with probe strand of final concentration of 100 pM under optimal conditions as mentioned above. Cell Culture. Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Walkersville, MD). M199 medium, heparin, gelatin, and endothelial cell growth supplement (ECGS) were purchased from Sigma. Fetal bovine serum (FBS) and penicillin-streptomycin (PS) were purchased from Invitrogen. Dulbecco’s modified Eagle medium (DMEM) and Roswell Park Memorial Institute medium (RPMI) were purchased from Gibco (Grand Island, NY). HUVEC were grown in M199 medium supplemented with 20% heat-inactivated FBS, 20 µg/mL ECGS, 90 µg/mL heparin, 1% PS in 75 cm2 culture flasks coated with 0.1% gelatin. HUVEC from passage 2-6 were used in this study. In addition, human hepatocellular carcinoma (HepG2) was cultured in DMEM supplemented with 10% FBS and 1% PS, and invasive breast ductal carcinoma (MCF-7) was cultured in RPMI supplemented with 10% FBS and 0.5% PS. All cells were maintained in a humidified incubator at 37 °C with 5% CO2 and 80% relative humidity. Total RNA Isolation. Total RNA of HUVEC, HepG2, and MCF-7 were extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Briefly, the sample was lysed and homogenized with TRIzol Reagent. Phase separation was followed by addition of chloroform and centrifugation. RNA in the aqueous phase was then recovered by precipitation with isopropyl alcohol. The RNA pellet was washed with 75% ethanol and finally redissolved in RNase-free water. The RNA quantity was determined by measuring optical density at 260 nm using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the RNA quality was assessed by performing agarose gel electrophoresis. Quantification of miR-21 in Cells by Quantitative RealTime PCR (qRT-PCR). Complementary DNA (cDNA) was generated from 10 ng of total RNA per 5 µL of gene specific reverse transcription (RT) reaction by using reagents from TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and mature hsa-miR-21-specific RT primer (5×) from TaqMan MicroRNA Assays (P/N: 4373090, Applied Biosystems). Each RT reaction contained 1 mM dNTPs, 50 U MultiScribe reverse transcriptase, 1× reverse transcription buffer, and 3.8 U RNase inhibitor as well as 1× specific RT primer. The reaction mixture was incubated in PTC-100 Programmable Thermal Controller (MJ Research, Inc., Waltham, MA) for 30 min at 16 °C, 30 min at 40 °C, 5 min at 85 °C, and then held at 4 °C. The cDNA sample was then amplified by PCR using TaqMan 2× Universal PCR Master Mix (No AmpErase UNG) (Applied Biosystems) and TaqMan Assay (20×) from TaqMan MicroRNA Assays (P/N: 4373090, Applied Biosystems). Each PCR reaction included 0.67 µL of RT product, 5 µL of TaqMan 2× Universal PCR Master Mix, No AmpErase UNG, and 0.5 µL of 20× TaqMan Assay (a mix preformulated miRNA-specific forward PCR primer, specific reverse PCR primer and miRNA-specific TaqMan MGB probe). The reaction mixture was then run at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min in Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

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Figure 1. Schematic illustration of the hybridization-based TIRFM assay for the detection of single miRNA molecules in solution.

Bio-Rad iCycler, Version 4.006 (Bio-Rad Laboratories, Inc., Hercules, CA). The PCR reactions were run along with no-template control and RT-minus control. Data were analyzed by iCycler iQ Optical System Software, Version 3.0a (Bio-Rad Laboratories), and the miRNA expression level was measured using threshold cycle (Ct) which is the cycle number at which the fluorescence generated within a reaction crosses the threshold. The Ct values were then converted to absolute amount using a standard curve of mature miR-21. Six independent experiments were performed, and each experiment was run in duplicate. Establishment of miR-21 Calibration by qRT-PCR. A mixture of synthetic RNA oligonucleotides from mirVana miRNA Reference Panel v9.1 (Applied Biosystems) was used to generate a standard curve for miR-21. The RNA oligonucleotides representing mature miR-21 ranging from 10-6 to 1 fmol were reverse transcribed, also using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and mature miR-21-specific TaqMan MicroRNA Assays (P/N: 4373090, Applied Biosystems). The PCR amplification of the cDNA was then performed using same materials as mentioned above. Three independent experiments were performed, and each experiment was run in duplicate. Calibration curve was shown in Supporting Information. Quantification of miR-21 in Cells by TIRFM. Total RNA of HUVEC, HepG2, and MCF-7 was diluted to 150 ng/µL with TNE buffer, respectively. Consequently, 7.5 µL of diluted total RNAs were spiked into a mixture standard solution of miR-21 of a final concentration of 0, 1, 2, 5, 10, and 15 pM and a LNA probe with a final concentration of 100 pM, and finally diluted with TNE buffer to a volume of 44 µL. The solution was incubated for 1 h at 52 °C and followed by addition of 1 µL of YOYO dye as mentioned previously. After equilibrium for 5 min, 10 µL (with 250 ng of total RNA) of solution was pipetted to coverslips for TIRF imaging and quantitation. The contents of miR-21 in the three cell lines were estimated by standard addition method and the value of miR-21 quantified by the TIRFM platform was compared with the outcome of the qRT-PCR method. Imaging System. An inverted Olympus IX-71 microscope (Olympus, Tokyo, Japan) was equipped with a high-numerical aperture 60× oil-immersion objective (1.45 NA, PlanApo, Olympus) as shown in Figure S1A. The sample coverslip was located under the fused-silica Isosceles Brewster Prism (CVI Melles Griot, Carksvadm, CA) and above the 60× objectives with immersion oil (η ) 1.52, Nonfluorescence, Olympus) in between. A 488 nm cyan laser (50 mW, CMA1-01983, Newport, NJ) was used as the excitation source. The laser beam was first filtered with neutral 6914

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density filter (FSR-OD 60, Newport), focused by cylindrical lens (focal length, 150 mm; CVI Melles Griot), and eliminated with the aid of pinholes before its entry to the prism with an incident angle of approximately 66°. Evanescent field was generated by the TIR of laser beam occurring at prism and was used to excite the fluorescently labeled hybrid molecules. A band-pass emission filter (D535/40 m Chroma Technology) was placed between the objective lens and the EMCCD to collect emitted photons. A uniphase mechanical shutter (model LS272, Vincent Associates, Rochester, NY) and a driver (model VMM-T1, Vincent Associates) were synchronized with the PhotonMax: 512B electron-multiplied CCD camera (EMCCD, Princeton Instruments, Princeton, NJ) in external synchronization mode and frame-transfer mode. The mechanical shutter blocked laser beam when the camera was off in order to reduce photobleaching. The ADC rate of the camera was 10 Hz, exposure time was 50 ms, and the multiplication gain was set at 4000, with the shutter driver set to 100 ms exposure and 100 ms delay. Typically, an image series of 10 sequential frames on 10 locations were acquired from a single slide. Images were obtained with the WinSpec/32 software (Version 2.5.22.0, Downingtown, PA) provided by Princeton Instruments. Data Analysis. All captured images were analyzed with a public-domain image-processing program Image J (version 1.43i, NIH, Bethesda, MD). A region of interest (ROI) with 200 pixelssquare at the center of the light spot with relatively even laser intensity was selected for single molecule counting. Intensity of images may also suffer while the shutter was triggered on or off. Five subframes (frame 3 to frame 7) of the image series were selected for analysis. The threshold for image acquirement was chosen at a value of three times the standard deviation of the mean intensity of the image. The image was then further processed with the Analyze Particles function in Image J to determine the number of single fluorescence particles computationally. The size of particles was set at 2-10 pixels to reduce false positive signals generated from noises. Number of spots in five frames was counted separately and summed up (i.e., accumulation of spots imaged within 250 ms). Consequently, the sum of spots from 10 image series of a single slide was averaged. All experiments were done in triplicate and the error bars of charts shown in the Results and Discussion refer to the standard error of mean of the triplicate experiments unless specified. RESULTS AND DISCUSSION In this study, the miRNA detection is based on the hybridization approach as illustrated in Figure 1. Complementary probes of oligonucleotides were hybridized with the target miR-21 in

solution under appropriate incubation conditions. Compared with surface-based hybridization that involves washing of excess reagents, solution-based hybridization offers the advantage of higher hybridization efficiency. The hybridized duplexes were labeled with fluorescent dye YOYO-1 iodide (YOYO) for the singlemolecule fluorescence detection. YOYO is an intercalating fluorescent dye which electrostatically binds to the backbone of oligonucleotide.41-43 The binding affinity of YOYO dyes on double-stranded oligonucleotide is very high (∼6 × 108 M-1) and there is a fluorescence intensity enhancement of approximately 400-fold upon binding to DNA.42 Since the binding mechanism is based on geometrical insertion, neither dye molecules nor the oligonucleotides have to be chemically modified during the labeling process. The detection of miRNA is, thus, straightforward. To improve the fluorescence signal intensity, the hybrids (∼20 bp) were labeled with YOYO in the ratio of 1 dye molecule/1 bp. After direct labeling of hybrids with YOYO, microliters of sample solution were sandwiched in a pair of precleaned coverslips with a solution depth of approximately 20 µm, and observed under EMCCD-TIRF microscope. The cleanness of the coverslips was found to be very significant in the assay as scattering and autofluorescence of any dirt and stains on glass surface may result in false positive signals. It is crucial to clean coverslips extensively before use. YOYO-labeled miRNA hybrids were visualized by a singlemolecule TIRFM imaging system (Figure S1A). A 488 nm laser was used to excite the bound YOYO dyes. The laser beam with an incident angle of approximately 66° was total-internal-reflected at the glass/solution interface. The thickness of evanescent field layer (EFL) generated by total internal reflection was calculated to be ∼190 nm by d ) λ/(2π(η22 sin2 θ - η12)1/2), where d is the penetration depth of the field, λ is the wavelength of the excitation light in vacuum, η1 and η2 are the refraction indices of the solution and glass slides, and θ is the angle of incidence. For more homogeneous exciting laser intensity, a central region of 200 × 200 pixels (53 × 53 µm2) of the EMCCD image was selected as the sampling area, and thus, the probe volume is estimated to be 0.54 pL. When 100 pM of miRNA hybrids was loaded on the coverslip, the theoretical number of observed hybrid molecules existing in the sampling region was 100 pM × (53 µm × 53 µm) × 190 nm × 6.02 × 1023 molecules/mol ) 33.44 Figure S1B shows a typical TIRFM image of miRNA hybrids acquired in single-molecule level. It is noted that molecules undergo random diffusional motion in a nonimmobilized system.45 The diffusion coefficient of the miRNA hybrids was calculated as 76 µm2 s-1 in bulk solution.46 However, it was showed that molecular diffusion rate is much slower at the glass/solution interface compared to the bulk because of the electrostatic interaction between molecules and macroscopic glass surface in microsized domain.45,47-49 The fluorescence signal generated (41) Cosa, G.; Focsaneanu, K. S.; McLean, J. R. N.; McNamee, J. P.; Scaiano, J. C. Photochem. Photobiol. 2001, 73, 585–599. (42) Gurrieri, S.; Wells, K. S.; Johnson, I. D.; Bustamante, C. Anal. Biochem. 1997, 249, 44–53. (43) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. 1992, 20, 2803–2812. (44) He, Y.; Li, H. W.; Yeung, E. S. J. Phys. Chem. B 2005, 109, 8820–8832. (45) Xu, X. H.; Yeung, E. S. Science 1997, 275, 1106–1109. (46) Zhdanov, V. P. Mol. BioSyst. 2009, 5, 638–643. (47) Xu, X. H. N.; Yeung, E. S. Science 1998, 281, 1650–1653.

Figure 2. Correlation between the number of observed miR-21 molecules and expected number of miR-21 in the sampling volume (0.54 pL). The number of observed miR-21 was corrected as the net number of hybrids (number of observed molecules - number of observed in blank). The slope of the correlation curve is 0.80, which indicates the assay has a hybridization efficiency of approximately 80%.

from the single molecules is spreaded as they diffuse and the size of the fluorescence spots in the image is larger than the physical size of molecules of interest (see also movie file in Supporting Information).45 Although molecules interact with the glass surface, nonspecific adsorption of hybrid molecules on the surface of glass slide was insignificant as both the oligonucleotides and the glass surface are highly negative-charged at pH 7.4. Figure S2 shows the histogram of residence time for each miRNA hybrids in 8 consecutive frames. Among the 114 molecules detected in the 8 frames, ∼ 80% of the molecules appeared and then disappeared in a single frame; 11%, 4%, and 4% of the molecules stayed at the same position for 2, 3, and 4 frames, respectively, and less than 1% retained for 7 frames and eventually desorbed from the surface. In the TIRFM image, each fluorescent spot was regarded as a single molecule as a linear correlation on the number of counted fluorescence spots and the expected number of miR-21 calculated from the corresponding concentration was established (R2 ) 0.991). Herein, hybridization is the main factor attributed to the differences in observed and expected number of molecules and its efficiency was approximated to be 80% from the slope of the plot as shown in Figure 2. Optimization of Hybridization Conditions. The stringency of hybridization governs the detection sensitivity and selectivity. It is crucial to optimize the hybridization conditions before performing further detection. The effects of ionic strength, selection of probes, and incubation time on the hybridization efficiency were evaluated. First, the effect of buffer ionic strength on hybridization efficiency was studied (Figure 3A). In general, hybridization affinity is improved at higher ionic strength as the electrostatic repulsions between the negatively charged oligonucleotides can be effectively shielded in the presence of salt. However, high ionic strength may also result in aggregation of molecules. The aggregates will be misinterpreted as an individual in SMD, and (48) Lyon, W. A.; Nie, S. M. Anal. Chem. 1997, 69, 3400–3405. (49) Isailovic, S.; Li, H. W.; Yeung, E. S. J. Chromatogr., A 2007, 1150, 259– 266.

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Figure 3. Optimization of miRNA hybridization conditions. (A) Effect of ionic strength adjusted by NaCl on hybridization efficiency (number of hybrid counts). (B) Performance of LNA, DNA, and RNA probe on hybridization with complementary miR-21 and negative control of miR214. (C) Effect of incubation time on hybridization efficiency (number of hybrid counts). The data depict the averages of three experiments, and the error bars are the standard error of mean of the three trials.

as a consequence, the number of counts drops. Here, we adjusted the ionic strength by varying the concentration of NaCl in 1× TrisNaCl-EDTA (TNE) buffer. Figure 3A shows the molecule counts as a function of the concentration of NaCl in the hybridization buffers for probes of locked nucleic acid (LNA), DNA, and RNA, 6916

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respectively, with the same concentration of the target miR-21. The experimental condition was considered optimum when the highest number of molecule counts was obtained; indicating that the largest number of individual hybrids was formed. A gradual increase in the number of hybrids was observed from buffer containing 0-250 mM NaCl and a decrease after the salt concentration exceeds. We analyzed the pixel size of each fluorescent spots of LNA/miRNA hybrids prepared in TE buffer with 250 and 500 mM NaCl, respectively, as shown in Figure S3. It is obvious that higher percentages of molecules with larger pixel sizes (size >4 pixels) were found in the series of 500 mM NaCl compared to that of 250 mM. This implies that increase in ionic strength would induce aggregation among hybrids and cause underestimation on the count of molecules in single molecule level. Thus, it is concluded that the optimal NaCl concentration was 250, 150, and 150 mM with respect to probes of LNA, DNA, and RNA. Oligonucleotide probe is commonly used as a reporter in hybridization-based nucleic acid detection. Conventional miRNA detection assays use DNA oligonucleotides as the capturing probe, although the stability of DNA-miRNA duplex is not high. Recently, several groups demonstrated the potential of LNAs as an alternative to DNA probes.8,9,50,51 LNA is a nucleic acid analogue known as a mimic of RNA that has high binding affinity to RNA molecules. The thermostability of the LNA-miRNA duplex is significantly higher than that of unmodified DNA probes.52-55 Consequently, both the detection sensitivity and hybridization discrimination efficiency are enhanced due to the increase in binding affinity and stability of LNA-miRNA complex. To assess the performance of different nucleic acid analogues of probes in the detection of miRNA, the hybridization affinities of LNA, DNA, and RNA probes toward (i) complementary miR-21 and (ii) negative control miR-214 (sequence showed in Experimental Section) in free solution were investigated. As shown in Figure 3B, LNA probe-based hybridization yields a 1.5-fold enhancement in the molecule counts compared with those of DNA and RNA probes, while the binding of LNA probes to negative control of miR-214 was maintained at low level. This indicates that LNA probe not only promotes higher capturing efficiency, but also displays a good mismatch discrimination capability. Incubation time also plays a role on the overall hybridization efficiency. Counts of hybrids obtained after incubation for 15, 30, 60, and 180 min were shown in Figure 3C. It was observed that the number of counts increased and reached maximum in the first 60 min incubation and then reduced after 3 h of incubation. The reduction in counts after prolonged incubation may be due to the structural conformation change of LNA strands as proven by MALDI-TOF mass spectrometry analysis (see Supporting (50) Castoldi, M.; Schmidt, S.; Benes, V.; Noerholm, M.; Kulozik, A. E.; Hentze, M. W.; Muckenthaler, M. U. RNA 2006, 12, 913–920. (51) Kloosterman, W. P.; Wienholds, E.; de Bruijn, E.; Kauppinen, S.; Plasterk, R. H. A. Nat. Methods 2006, 3, 27–29. (52) Bondensgaard, K.; Petersen, M.; Singh, S. K.; Rajwanshi, V. K.; Kumar, R.; Wengel, J.; Jacobsen, J. P. Chem.sEur. J. 2000, 6, 2687–2695. (53) Braasch, D. A.; Corey, D. R. Chem. Biol. 2001, 8, 1–7. (54) Nielsen, K. E.; Rasmussen, J.; Kumar, R.; Wengel, J.; Jacobsen, J. P.; Petersen, M. Bioconjugate Chem. 2004, 15, 449–457. (55) Petersen, M.; Nielsen, C. B.; Nielsen, K. E.; Jensen, G. A.; Bondensgaard, K.; Singh, S. K.; Rajwanshi, V. K.; Koshkin, A. A.; Dahl, B. M.; Wengel, J.; Jacobsen, J. P. J. Mol. Recognit. 2000, 13, 44–53.

Figure 4. Standardization curve for the quantification of miR-21. Different concentrations of miR-21 were hybridized with 100 pM LNA probe in solution at 52 °C for 1 h. The data depict the averages of three experiments, and the error bars are the standard error of mean of the three trials.

Information, Figure S4). As a result, the hybridization time was set to 60 min for later experiments in this study.

Quantification of Synthetic miR-21. Under the optimal hybridization conditions of miRNAs as discussed above, a calibration plot of molecules counts as a function of the target miR-21 concentration was constructed. Synthetic miRNA of 0-100 pM was hybridized with 100 pM LNA probes in solution and labeled with YOYO. By single-molecule counting on the series of images acquired, a plot of the number of molecule counts as a function of the concentration of synthetic miR-21 was obtained with a coefficient of determination of 0.991 (Figure 4). The detection limit of the assay was estimated to be 5 pM (i.e., 50 amol in 10 µL of sample). The ultimate theoretic limit of single molecule detection is to detect a sole molecule in the bulk sample solution sandwiched between the glass slides. Ideally, the theoretic limit is calculated to be 170 zM, with a single target molecule in 10-µL sample solution. To achieve such an ultimate theoretic limit requires sufficiently long sampling time until the single target molecule enters the probe volume (0.54 pL) by chance and gets excited and detected. Otherwise, regardless of the sampling time, the concentration of sample solution should be ∼3 pM, so that a single molecule always locates in the probe volume. To improve the detection limit, several factors including the probe volume, viscosity of sample solution, sampling time, photostability, and

Figure 5. Quantification of miR-21 contents in total RNA of (A) MCF-7, (B) HepG2, and (C) HUVEC cells by standard addition methods with TIRFM. Synthetic miR-21 was spiked into matrix of total RNA and LNA probe. The data depict the averages of three experiments, and the error bars are the standard error of mean of the three trials. (D) Comparison between the miR-21 contents in total RNA of HUVEC, HepG2, and MCF-7 cells determined by qRT-PCR assay and single-molecule TIRFM assay. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

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quantum efficiency of fluorescence dyes may be optimized as reported by Nie and co-workers.56 Quantification of miR-21 in Normal and Cancerous Cell Lines. MCF-7 (Invasive breast ductal carcinoma cell line) and HepG2 (human hepatocarcinoma cell line) are regarded as a common breast and liver cancer cell line model, respectively, while HUVEC (human umbilical vein endothelial cells) is regarded as normal cell line. Herein, we employed the single-molecule detection assay to quantify differential-expressing miRNAs in normal and cancerous cell lines. Briefly, total RNA of each cell line was extracted prior to the direct miRNA detection. The YOYO dye has no selectivity toward oligonucleotides, mRNAs, tRNAs, and other small RNAs in such environment. To eliminate the signals drawn from the complex matrixes without supplementary pretreatments on the cell samples, standard addition method is adopted in the manner that synthetic target miR-21 is spiked to the mixture of total RNA sample and probes. The original concentration of miR-21 in the sample of total RNA of each cell lines can eventually be obtained by extrapolation of the calibration curve. Three independent standardization curves were prepared for the three cell lines and the amounts of miR-21 in each of the cell line were determined. All three calibration curves showed strong correlation between the numbers of counted molecules and the miRNA concentration with the coefficients of determination for all three of them greater than 0.997 (Figure 5A-C). Since the hybridization efficiency was ∼80% as mentioned previously, the concentration of miR-21 in each cell line estimated by standard addition method was multiplied by the efficiency conversion factor of 1.25 such that the actual miR-21 contents in total RNA can be obtained. The contents of miR-21 in total RNA of each cell line were found to be 0.92, 0.43, and 0.32 amol/ng for MCF-7, HepG2, and HUVEC, respectively. For the purpose of result validation, we quantified the content of miR-21 in the three cell lines using the same batch of cells by qRT-PCR method. Quantitative RT-PCR is a technique commonly adopted as a standard method for miRNA profiling. It is superior to other detection assays for its high specificity and minute amounts of starting materials used in the detection. Amplification steps however are involved and it takes approximately 5 h for the whole process. The output of qRT-PCR is usually expressed in terms of fold-change and so the raw data is semiquantitative. Additional calibration curve has to be established for quantitation purpose. To compare, our SMD detection assay is relatively rapid as it takes only 1 h of sample incubation and promptly followed by microscopic detection. The result is quantitative and obtained by applying standard addition methods. The standardization curve

by qRT-PCR is shown in the Supporting Information (Figure S5). Figure 5D displays the contents of miR-21 in MCF-7, HepG2, and HUVEC cells determined by the SMD assays and those by qRTPCR with calibration standardization. The SMD result agrees very well with the outcome of qRT-PCR. The high correlation with the accredited qRT-PCR methods demonstrated that the pretreatmentfree SMD system developed here is of high potential in profiling expression of miRNAs in different cell lines and thus applicable in early cancer diagnosis.

(56) Nie, S. M.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 2849–2857.

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CONCLUSIONS We developed a direct and amplification-free quantitative assay of single miRNA molecules using solution-based hybridization approach and fluorescence-based detection with TIRFM. This assay is straightforward, rapid, and highly sensitive. The fluorescent hybrids diffuse randomly in the refined detection volume. Improvement on the limit of detection could be achieved by increasing sampling time or immobilizing target fluorescent hybrids onto the coverslips. As a proof of concept, the content of miR-21 were determined in cancerous MCF-7 and HepG2 and noncancerous HUVEC cell lines and the result agreed very well with that of conventional qRT-PCR. Both the success in discriminating differentially expressing miR-21 in different cell lines and the high correlation with the conventional detection method justified the potential of the system in application for future early cancer diagnosis. ACKNOWLEDGMENT This work was fully supported by the Faculty Research Grant of Hong Kong Baptist University (FRG/07-08/II-68) and grant from the University Grants Council of the Hong Kong Special Administrative Region, China (HKBU I/06C). We thank Dr. C. K. C. Wong from the Department of Biology of HKBU for providing the MCF-7 cells. SUPPORTING INFORMATION AVAILABLE Additional information includes video of miRNA diffusing in probe volume, adsorption time analysis of single hybrid molecules, pixel size analysis of hybrids prepared in TE buffer containing 250 and 500 mM NaCl, MALDI-TOF mass spectrum of LNA strands, calibration curve of qRT-PCR. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 30, 2010. Accepted July 14, 2010.