Article pubs.acs.org/ac
High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification Haiyun Liu,‡ Lu Li,‡ Lili Duan, Xu Wang, Yanxia Xie, Lili Tong, Qian Wang, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan, 250014, China S Supporting Information *
ABSTRACT: In this paper, a padlock probe-based exponential rolling circle amplification (P-ERCA) assay is developed for highly specific and sensitive detection of microRNA (miRNA). The padlock probe is composed of a hybridization sequence to miRNA and a nicking site for nicking endonuclease. Using the miRNA as a template, specific ligation to the padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as triggers in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate the rolling circle amplification reaction, and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of the padlock probe. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to single−nucleotide difference discrimination between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics.
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detection can reach the single molecule level.23 However, it requires precise control of temperature cycling for successful amplification, and the short length of miRNAs makes their experimental design very sophisticated.23−26 Except these standard methods for miRNA detection mentioned above, various new strategies have been developed to improve the detection sensitivity and adaptability, such as nanoparticalbased assay,27−30 bioluminescence-based assay,31 modified invader assay,32 ribozyme-based assay,33 sequencing-based assay,34 EXPAR assay,35 strand displacement assay,36 and hairpin-based amplification.37 Among these methods, rollingcircle amplification (RCA) has become increasingly popular in miRNA detection due to its simplicity, specificity, and high sensitivity.38−40 After RCA-based miRNA detection was first reported,38 several novel strategies were developed to improve the specificity and sensitivity of this method by introducing a second primer,39 a dumbbell-shaped DNA probe,40,41 DNAzyme,42 or encoded gel microparticles.43 Recently, a primer generation-rolling circle amplification (PG-RCA) has been reported.44,45 PG-RCA is a process including a cascade reaction of linear rolling circle amplification and nicking reactions. In
icroRNAs (miRNAs) are short, endogenous, noncoding RNA of about 18−24 nucleotides (nt) that play important roles in normal and pathologic processes. They regulate gene activity and act to promote or repress cell proliferation, migration, and apoptosis.1−5 Recent studies have found that some miRNAs have altered expression in cancer cells; aberrant expression of miRNAs is associated with cancer initiation, tumor stage, and tumor response to treatments.5,6 Nowadays, miRNAs have been regarded as biomarkers and therapeutic targets in cancer treatment.1,4,7−11 So, effective detection of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in biomedical research and clinical diagnosis. However, it is difficult to analyze the miRNAs because of their unique characteristics, including their small size, sequence homology among family members, and low abundance in total RNA samples.12 So, strategies for specific, especially sensitive quantitive detection of miRNAs are in urgent need. Recently, many different methods have been used to profile miRNAs expression. Northern blot is widely used to visualize specific miRNA,13−16 but it requires large amounts of the sample, and the sensitivity is not satisfied.12 Microarray technology offers a way to analyze a little volume and multiple samples simultaneously. Nevertheless, its sensitivity and specificity should also be improved.17−22 RT-qPCR has been proposed for miRNA analysis, and the sensitivity of miRNA © 2013 American Chemical Society
Received: June 8, 2013 Accepted: July 16, 2013 Published: July 16, 2013 7941
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electrophoresis was conducted using a DYCZ-24DN Electrophoresis Cell (LIUYI, Beijing, China) and GelDoc-It Imaging Systems (UVP, Cambridge, U. K.). The fluorescent spectra were measured using a Cary Eclipse Fluorescence Spectrophotometer (Varian, CA). Ligation Reactions. The ligation reaction was carried out with a 10 μL reaction mixture containing 1 × ligation buffer [40 mM Tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 500 mM ATP (pH 7.8)], 2 U of T4 RNA ligase 2, 2 μL of the padlock probe, and 2 μL of miRNA. Before adding T4 RNA ligase 2 and the ligation buffer, the oligonucleotide mixture was denatured at 65 °C for 3 min and cooled slowly to room temperature over a 10 min period. After annealing, T4 RNA ligase 2 and ligation buffer were added to the mixture and incubated at 37 °C for 2 h. RCA Reactions. The amplification reaction was conducted at 30 °C in a 20 μL reaction mixture containing 10 μL of ligation reaction products, 20 mM Tris−HCl buffer (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 6 mM MgSO4, 400 μM each dNTP, 0.1% Triton X-100, 0.4 U Phi29 DNA polymerase, and 1 U Nb.BbvCI. Gel Electrophoresis Analysis. P-ERCA product was analyzed with 16% urea denaturing polyacrylamide gel electrophoresis (PAGE). The gel was carried in 1× electrophoresis Tris-borate-EDTA (TBE) at 100 V for 10 min, and stained with SYBR Green I for 15 min. The imaging of the gel was performed using the UVP GelDoc-It Imaging Systems. Measurement of Fluorescent Spectra. The 6 μL PERCA amplification product was mixed with 4 μL 20 × SYBR Green I and diluted to a final volume of 600 μL with 10 mM PBS (pH 7.4). The fluorescent spectra were measured using a Cary Eclipse Fluorescence Spectrophotometer. The excitation wavelength was 480 nm, and the spectra were recorded between 520 and 650 nm. The fluorescence emission maximum was at 528 nm. Cell Lysis and RNA Preparation. The human lung cells (A549) were cultured according to the instructions of the American Type Culture Collection. Cells were grown in RPMI 1640 (Hyclone, penicillin 100 U/mL, streptomycin 100 μg/ mL) plus 10% fetal bovine serum (FBS, Gibco) and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells were collected and centrifuged at 3000 rpm for 5 min in a culture medium, washed once with PBS buffer, and then spun down at 3000 rpm for 5 min. The cell pellets were suspended in 600 μL of lysis solution. Total RNA was extracted from human lung cells using the mirVana miRNA Isolation Kit according to the manufacturer’s procedures. The RNA concentration was determined to be 3.9 μg/mL from the UV vis absorption at 260 nm. The sample of let-7a in these cells was diluted, then analyzed with the proposed miRNA assays.
contrast with conventional linear rolling circle amplification, the amplification was in an exponential mode. The remarkable sensitivity may offer great possibility for low-abundance miRNA detection. But due to the high sequence homology among miRNA family members and small size, there’s still a great challenge for specific detection of miRNA, especially for the discrimination of single-nucleotide difference within the same and short lengths. Herein, we developed a padlock probe-based exponential rolling circle amplification (P-ERCA) assay for highly specific and ultrasensitive detection of miRNA. After a padlock probe was designed, reasonable, specific ligation and circulation with the miRNA as a template was achieved under isothermal conditions. The ligation reaction and the target-dependent circularization of the padlock probe could improve the specificity of the miRNA assay effectively. The exponential amplification allowed quantification of miRNA with a remarkable sensitivity of 0.24 zmol. The miRNA analysis in human lung cells was also performed, indicating that this PERCA strategy will become a specific and ultrasensitive miRNA quantification method in medical research and early clinical diagnostics.
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EXPERIMENTAL SECTION Materials and Apparatus. Oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The miRNA, diethyprocarbonated (DEPC)-treated deionized water, deoxynucleotides (dNTPs), TE buffer, PBS, and ribonuclease inhibitor were obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). T4 RNA ligase 2, T4 DNA ligase, Phi29 DNA polymerase, and Nb.BbvCI were purchased from New England Biolabs (Ipswich, MA). SYBR Green I was purchased from Xiamen Zeesan Biotechnology Co., Ltd. (Xiamen, China). Fetal bovine serum was purchased from Gibco (Carlsbad, CA). The RPMI 1640 and MirVana miRNA Isolation Kit were purchased from Life Technologies (Carlsbad, CA). Before use, the oligonucleotides and miRNA were diluted to appropriate concentrations with diethprocarbonated (DEPC)-treated water, then separated into 20 μL centrifugal tubes. All of the oligonucleotides and miRNA were purified by HPLC. DEPCtreated deionized water was used in all experiments. The sequences of the oligonucleotiedes and miRNAs were listed in Table 1. An isothermal amplification reaction was performed on a Veriti 96 Well Thermal Cycler (Applied Biosystems, CA). Gel Table 1. Sequences of oligonucleotides and miRNAsa name
sequence (5′-3′)
padlock probe I
5′-phosphate-CTA CTA CCT CAT TTG CAT TTC AGT TTA CCT CAG CGC ATT TCG CAA TTT TAA CTA TAC ACC3′ 5′-phosphate-CTA CTA CCT CAC CTC AGC AAC TAT ACA ACC TAC TAC CTC ACC TCA GCA ACT ATA CAA CCT ACT ACC TCA CCT CAG CAA CTA TAC AAC-3′ 5′-UGA GGU AGU AGG UUG UAU AGU U-3′ 5′-UGA GGU AGU AGG UUG UGU GGU U-3′ 5′-UGA GGU AGU AGG UUG UAU GGU U-3′ 5′-UAG CUU AUC AGA CUG AUG UUG A-3′
padlock probe II let-7a let-7b let-7c miR-21
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RESULTS AND DISCUSSION Principle of P-ERCA assay. The strategy for miRNA detection on the basis of the padlock probe-based exponential rolling circle amplification (P-ERCA) is shown in Scheme 1. The padlock probe is composed of a hybridization sequence to miRNA (black) and a nicking site for nicking endonuclease (red). The 5′- and 3′-termini of the padlock probe are designed to be complementary to the miRNA target. The padlock probe can be ligated specifically and circularized with the miRNA as a template in the presence of T4 RNA ligate 2. Once miRNA and the padlock probe form a complex, Phi29 DNA polymerase synthesizes a long concatenated sequence copy of the padlock
a
The bold characters indicate recognition sequences of Nb.BbvCI. The underlined bases are the different bases between let-7b, let-7c, and let-7a. 7942
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ERCA pathway of cleavage by nicking endonuclease and production of multiple DNA products, denaturing polyacrylamide gel electrophoresis was also performed. The P-ERCA after triggering by the let-7a target was conducted, and productions were analyzed using 16% denaturing polyacrylamide gel electrophoresis. Lane 1 in the inset of Figure 1 shows the amplification products by P-ERCA in the presence of let-7a; the wide bands of lane 1 could be attributed to the multiple components of the cleavage. This is probably because the LRCA product from padlock probe II has nicking sites every 22 bases and even the ladder products could look like smear.44 When the reaction was conducted in the absence of let-7a, there is no product observed (lane 0 in the inset of Figure 1), indicating no reaction occurs also. Specificity of the Assay. It is a great challenge to carry out the miRNA assay with high specificity due to the high sequence homology among family members and small size of miRNA. For example, members of the let-7 miRNA family differ by only one or two nucleotides in sequence with the same length (only 22 bases). In this assay, a padlock probe was introduced to improve the selectivity. In the padlock probe, two targetcomplementary segments are present at opposite ends of a linear DNA probe molecule. Upon hybridization to the target miRNA, the ends of the padlock probe are brought in contact, and the probe is circularized by enzymatic ligation of the ends, trapping the probe molecule at the site of hybridization. Due to the strict requirement for coincident hybridization to two target segments, target miRNA was recognized with very high specificity; even single-nucleotide variants of sequences could be distinguished using the padlock probe.46−50 Here, a circular probe was used to be compared with the padlock probe in the specificity evaluation of P-ERCA. Using the circular probe perfectly complementary to let-7a, fluorescence intensity produced by let-7a is only 1.6-fold of that produced by let-7c. When a padlock probe was used, the fluorescent signal produced by let-7a is approximately 6.2-fold higher than that of let-7c (Figure 2A), revealing that the padlock probe has higher specificity to discriminate a single-nucleotide difference between let-7a and let-7c compared with a circular probe. At the same time, the molecular dynamics simulation had been carried out to study the specificity of the two probes and the corresponding DNA-RNA binding free energy was shown in Figure 2B. It is clearly seen that the binding free energy of let7a was only 1.1-fold that of let-7c in the circular probe, while the ratio of binding free energy of let-7a to let-7c was about 1.5fold in the padlock probe. This was consistent with the experimentally observed results. The results illustrate that the ligation reaction allows an efficient, specific assay for discriminating single-nucleotide differences between miRNAs. The specificity of P-ERCA is also affected by the ligase for sealing the termini of the padlock probe. Both T4 DNA ligase and T4 RNA ligase 2 are generally used as an efficient catalyst of RNA ligation in RNA/DNA hybrids. The let-7a and let-7c were detected using T4 DNA ligase and T4 RNA ligase 2, respectively. The results shown in Figure 2C indicated though the fluorescent intensity produced by T4 RNA ligase 2 was lower than that of T4 DNA ligase, T4 RNA ligase 2 exhibited higher specificity than T4 DNA ligase in this assay, and the result was consistent with that reported by other literature reports.39 After the padlock probe and T4 RNA ligase 2 were used, the specificity of the proposed P-ERCA reaction was evaluated. Three members of the let-7 family (let-7a, let-7b, and let-7c), with only one- or two-nucleotides differences and miR-
Scheme 1. Scheme for miRNA Detection with the Padlock Probe-Based Exponential Rolling Circle Amplification (PERCA) Reactiona
a
The reaction involves three principal steps: (1) The padlock probe is ligated specifically and circularized with the miRNA as template in the presence of T4 RNA ligate 2. (2) A long concatenated sequence copy of the padlock probe is synthesized by Phi29 DNA polymerase. (3) Multiple padlock probes can be hybridized to the DNA product. Nicking endonuclease recognizes the nicking sites and cleaves sequences. Multiple triggers are produced and initiate a new reaction cycle. Through multiple reaction cycles, an exponential amplification for a small amount of miRNA is achieved.
probe through linear rolling circle amplification. Next, multiple padlock probes can be hybridized to multiple sites of the LRCA DNA product, and nicking endonuclease recognizes the sites and cleaves sequences of double strand formation, producing multiple short DNA products as new triggers to initiate multiple reaction cycles until some of the reaction components, most likely dNTP substrates, are depleted. Hence, LRCA and cleavage can be repeated continuously in cycles, conventional LRCA is converted to an exponential amplification, and a highly sensitive assay for miRNA can be achieved. The P-ERCA reaction was further confirmed by the measurement of fluorescent spectra and gel electrophoresis. As shown in Figure 1, the fluorescence intensity gradually increases in the presence of let-7a (curve b), indicating that let7a initiates the P-ERCA reaction and produces a large number of DNA products. On the contrary, the fluorescence intensity is faint and unchanged in the control reaction without let-7a (curve a), indicating that no reaction occurs. To verify the P-
Figure 1. Fluorescence intensity of amplification products by P-ERCA in the absence (a) and presence (b) of let-7a. Inset: Denaturing polyacrylamide gel electrophoresis (16%) of amplification products by the P-ERCA reaction in the absence (a) and presence (b) of let-7a. The products were stained with SYBR Green I. The marker was indicated by M. The reaction solution contained a 3 nM padlock probe and 18 zmol let-7a. The ligation reactions were performed at 37 °C for 2 h, and the amplification reactions were performed at 30 °C for 6 h. 7943
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Figure 2. (A) Comparison of the fluorescence intensity produced by let-7a and let-7c when a circular probe or a padlock probe was used, respectively. (B) Comparison of the binding free energy of let-7a and let-7c with circular probe and padlock probe. (C) Comparison of the fluorescence intensity produced by let-7a and let-7c when T4 DNA ligase or T4 RNA ligase 2 was used respectively. (D) Comparison of the fluorescence intensity produced by let-7a, let-7b, let-7c, and miR-21 using a padlock probe perfectly complementary to let-7a, where F and F0 are fluorescence intensities of amplification products by P-ERCA in the presence and absence of let-7a, respectively. The reaction solution contained 3 fM padlock probe and 18 zmol let-7a. The ligation reactions were performed at 37 °C for 2 h, and P-ERCA reactions were performed at 30 °C for 6 h. Error bars were estimated from three replicate measurements.
21, are chosen as the detection model. As shown in Figure 2D, the fluorescence signal produced by let-7a which is perfectly complementary to the padlock probe reaches 219, which is 39fold more than that of mir-21, 12-fold more than that of let-7b, and 6.2-fold more than that of let-7c, even though there is only a single-nucleotide difference between let-7a and let-7c, suggesting the high specificity of our strategy for miRNA detection. Optimization of Padlock Probe Sequences. The padlock probe used to ligate miRNA and initiate multiple reaction cycles carries two types of sequences for signal amplification: a hybridization sequence to the miRNA and a nicking site. Precise design for sequences is important in the amplification efficiency. Here, two padlock probes (padlock probes I and padlock probes II) were designed and amplification efficiency was compared. Padlock probe I used in the reaction contains only one complementary segment to miRNA and one nicking site for Nb.BbvCI, while padlock probe II contains three complementary segments and three nicking sites (Table 1). The P-ERCA reaction for let-7a detection was achieved using the two probes, results were shown in Figure 3. The fluorescence intensity produced by padlock probe II was 3.2-fold of that produced by padlock probe I. The increased complementary segments and nicking sites could increase the efficiency of LRCA and nicking reaction in each reaction cycle, producing more short DNA products as triggers and leading to multiple signal amplification.
Figure 3. Comparison of amplification efficiency with padlock probes I and II, where F and F0 are fluorescence intensities of amplification products by P-ERCA in the presence and absence of let-7a, respectively. The reactions were conducted with 3 fM padlock probe and 18 zmol let-7a. The ligation reactions were performed at 37 °C for 2 h, and amplification reactions were performed at 30 °C for 6 h. Error bars were estimated from three replicate measurements.
Optimization of P-ERCA Reaction Conditions. Several detection conditions such as the concentrations of dNTP substrates, Phi29 DNA polymerase, and nicking enzyme Nb.BbvCI; the time and temperature of ligation; and amplification were further optimized to improve the detection sensitivity. The final fluorescence was measured in the presence 7944
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Figure 4. The relationship between the fluorescence intensity and the concentration of dNTP (A), Phi29 DNA polymerase (B), Nb.BbvCI (C), the time of ligation and amplification (D), and the temperature of ligation and amplification (E), where F and F0 are fluorescence intensities of amplification products by P-ERCA in the presence and absence of let-7a, respectively. The concentration of let-7a was 18 zmol. The error bar represents the standard deviation of three measurements.
sensitivity, 400 μM dNTP, 0.4 U Phi29 DNA polymerase, and 1 U Nb.BbvCI were selected, respectively. The changes of fluorescence intensity with the time of ligation and amplification were also investigated (Figure 4D); 2 and 6 h were selected as the optimal ligation time and amplification time for miRNA detection. Several temperatures of ligation (35, 37, and 39 °C) and exponential rolling circle amplification (30 and 37 °C) were compared to obtain higher detection sensitivity (Figure 4E). Finally, 37 and 30 °C were considered to be the optimal ligation temperature and amplification temperature, respectively. Sensitivity of the Assay. Under the optimized concentrations, the let-7a was detected to evaluate the sensitivity of the proposed strategy. The fluorescent intensities of products via the P-ERCA reaction with different amounts of let-7a were measured (Figure 5), and there was a remarkable increase with
of 18 zmol let-7a. To investigate the influence of the concentration of dNTP, 100 μM, 200 μM, 400 μM, 600 μM, and 800 μM dNTP were used. As shown in Figure 4A, as the concentration of dNTP increased, the fluorescence intensity increased gradually until 400 μM dNTP was used. The concentration of Phi29 DNA polymerase is another factor which affects the amplification reaction. When different concentrations of Phi29 DNA polymerase were used, the fluorescence signals were analyzed, and the dates are shown in Figure 4B. The produced fluorescence signals increased with increasing concentration of Phi29 DNA polymerase until the concentration reached 0.4 U. The effect of the amount of Nb.BbvCI on the assay was assessed also. The dates were shown in Figure 4C. Though the amount of nicking enzyme was changed from 0.2 U to 2 U, there was little change in the fluorescence signals. In order to obtain optimal detection 7945
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calculated at 5.05 × 109 copies/μg, which was in good agreement with those obtained in previous studies.39,41
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CONCLUSIONS In summary, a highly specific and ultrasensitive isothermal detection of label-free miRNA is achieved based on the PERCA reaction. This reaction is initiated by the target miRNA and catalyzed by DNA products generated and accumulated during the reaction. Unlike conventional nucleic-acid amplification reactions such as the polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or nonspecific amplification. This experiment is a simple one. With the exponential amplification, fluorescence signals can be sensitively detected with a remarkable sensitivity of 0.24 zmol. Moreover, using the reasonable designed padlock probe and T4 RNA ligase 2 in the specific ligation, high specificity was achieved, even single− nucleotide difference can be discriminated. In addition, the proposed strategy has successfully achieved the detection of let7a in total RNA sample extracted from human lung cells. The results indicate that the proposed P-ERCA strategy holds great potential for further application in biomedical research and early clinical diagnostics.
Figure 5. The relationship between the fluorescent response and the amount of target miRNA (let-7a). The reactions were conducted with 3 fM padlock probe and miRNA from 0.3 zmol to 18 zmol.
the increased amount of let-7a. A good linearity was obtained in 3 orders of magnitude from 0.3 zmol to 18 zmol miRNA. The correlation equation was F − F0 = 28.26 + 10.91 A (F and F0 represent fluorescence intensities of amplification products by P-ERCA in the presence and absence of let-7a; A represents the amount of let-7a, zmol) with a correlation coefficient r = 0.9919. A relative standard deviation (RSD) of 4.2% for 11 repetitive measurements of 6.0 zmol let-7a was obtained, providing a good reproducibility of this miRNA assay. The detection limit was estimated to be 0.24 zmol (3σ, n = 11). The proposed assay has achieved one of the most sensitive approaches for miRNA detection compared to other reported methods (Table 2). The low detection limit allows ultrasensitive accurate quantitation of miRNA at low concentrations, which is of great significance in the early diagnosis of diseases.
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S Supporting Information *
Process of molecular dynamics simulation, calculations of binding free energies, and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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methods
dynamic range
references
Branched RCA CC-SDR D-RCA encoded gel microparticles-RCA P-ERCA EXPAR stem-loop RT-PCR
6 amol 136 zmol 20 zmol 15 zmol 0.24 zmol 0.1 zmol 7 copies
3 3 8 6 3 10 7
39 36 40 43 this work 35 23
AUTHOR INFORMATION
Corresponding Author
*Tel.: (86)531 86180010. Fax: (86)531 86180017. E-mail:
[email protected].
Table 2. The Comparison for Existing miRNA Amplification Methods detection limit
ASSOCIATED CONTENT
Author Contributions ‡
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21227005, 21035003, 21205074, 31200545), Specialized Research Fund for the Doctoral Program of Higher Education of China (20113704130001), Program for Changjiang Scholars and Innovative Research Team in University, the Shandong Distinguished Middle-Aged and Young Scientist Encourage and Reward Foundation (BS2012SW022), and Key Project of Chinese Ministry of Education (212102).
Real Sample Assay. P-ERCA was further applied to quantify the amount of let-7a in the total RNA sample that was extracted from human lung cells. The concentration of let-7a in human lung cells was determined using the standard addition method using synthetic let-7a as the standard. The human lung total RNA (3.9 μg/mL) extracted from human lung cells was diluted to 0.78 ng/mL with RNase free TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 7.5). Aliquots of the diluted RNA sample (1 μL) were spiked with standard solutions containing synthetic let-7a at concentrations of 0 zmol, 0.6 zmol, 1.8 zmol, 3.0 zmol, 4.2 zmol, 6 zmol, 9 zmol, 12 zmol, and 18 zmol, respectively. Then, P-ERCA and fluorescence detection were performed under the same conditions as described in the Experimental Section. The results are shown in Figure S1. The content of let-7a in the human lung total RNA sample was
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REFERENCES
(1) Sawyers, C. L. Nature 2008, 452, 548−552. (2) Lu, J.; Getz, G.; Miska, E. A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebert, B. L.; Mak, R. H.; Ferrando, A. A.; Downing, J. R.; Jacks, T.; Horvitz, H. R.; Golub, T. R. Nature 2005, 435, 834−838. (3) Ventura, A.; Jacks, T. Cell 2009, 136, 586−591. (4) Rossi, J. J. Cell 2009, 137, 990−992. (5) Johnson, S. M.; Grosshans, H.; Shingara, J.; Byrom, M.; Jarvis, R.; Cheng, A.; Labourier, E.; Reinert, K. L.; Brown, D.; Slack, F. J. Cell 2005, 120, 635−647.
7946
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Article
(39) Cheng, Y.; Zhang, X.; Li, Z.; Jiao, X.; Wang, Y.; Zhang, Y. Angew. Chem., Int. Ed. 2009, 48, 3268−3272. (40) Zhou, Y.; Huang, Q.; Gao, J.; Lu, J.; Shen, X.; Fan, C. Nucleic Acids Res. 2010, 38, e156. (41) Bi, S.; Cui, Y.; Li, L. Anal. Chim. Acta 2013, 760, 69−74. (42) Wen, Y.; Xu, Y.; Mao, X.; Wei, Y.; Song, H.; Chen, N.; Huang, Q.; Fan, C.; Li, D. Anal. Chem. 2012, 84, 7664−7669. (43) Chapin, S. C.; Doyle, P. S. Anal. Chem. 2011, 83, 7179−7185. (44) Murakami, T.; Sumaoka, J.; Komiyama, M. Nucleic Acids Res. 2009, 37, e19. (45) Murakami, T.; Sumaoka, J.; Komiyama, M. Nucleic Acids Res. 2012, 40, e22. (46) Nilsson, M.; Barbany, G.; Antson, D. O.; Gertow, K.; Landegren, U. Nat. Biotechnol. 2000, 18, 791−793. (47) Qi, X.; Bakht, S.; Devos, K. M.; Gale, M. D.; Osbourn, A. Nucleic Acids Res. 2001, 29, e116. (48) Baner, J.; Nilsson, M.; Mendel-Hartvig, M.; Landegren, U. Nucleic Acids Res. 1998, 26, 5073−5078. (49) Nilsson, M.; Krejci, K.; Koch, J.; Kwiatkowski, M.; Gustavsson, P.; Landegren, U. Nat. Genet. 1997, 16, 252−255. (50) Nilsson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.; Chowdhary, B. P.; Landegren, U. Science 1994, 265, 2085−2088.
(6) He, L.; Thomson, J. M.; Hemann, M. T.; Hernando-Monge, E.; Mu, D.; Goodson, S.; Powers, S.; Cordon-Cardo, C.; Lowe, S. W.; Hannon, G. J.; Hammond, S. M. Nature 2005, 435, 828−833. (7) Tricoli, J. V.; Jacobson, J. W. Cancer Res. 2007, 67, 4553−4555. (8) Ryan, B. M.; Robles, A. I.; Harris, C. C. Nat. Rev. Cancer 2010, 10, 389−402. (9) Mitchell, P. S.; Parkin, R. K.; Kroh, E. M.; Fritz, B. R.; Wyman, S. K.; Pogosova-Agadjanyan, E. L.; Peterson, A.; Noteboom, J.; O’Briant, K. C.; Allen, A.; Lin, D. W.; Urban, N.; Drescher, C. W.; Knudsen, B. S.; Stirewalt, D. L.; Gentleman, R.; Vessella, R. L.; Nelson, P. S.; Martin, D. B.; Tewari, M. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 10513−10518. (10) Cao, Y.; DePinho, R. A.; Ernst, M.; Vousden, K. Nat. Rev. Cancer 2011, 11, 749−754. (11) Calin, G. A.; Croce, C. M. Nat. Rev. Cancer 2006, 6, 857−866. (12) Cissell, K. A.; Shrestha, S.; Deo, S. K. Anal. Chem. 2007, 79, 4754−4761. (13) Valoczi, A.; Hornyik, C.; Varga, N.; Burgyan, J.; Kauppinen, S.; Havelda, Z. Nucleic Acids Res. 2004, 32, e175. (14) Varallyay, E.; Burgyan, J.; Havelda, Z. Nat. Protoc. 2008, 3, 190− 196. (15) Varallyay, E.; Burgyan, J.; Havelda, Z. Methods 2007, 43, 140− 145. (16) Pall, G. S.; Hamilton, A. J. Nat. Protoc. 2008, 3, 1077−1084. (17) Babak, T.; Zhang, W.; Morris, Q.; Blencowe, B. J.; Hughes, T. R. RNA 2004, 10, 1813−1819. (18) Nelson, P. T.; Baldwin, D. A.; Scearce, L. M.; Oberholtzer, J. C.; Tobias, J. W.; Mourelatos, Z. Nat. Methods 2004, 1, 155−161. (19) Thomson, J. M.; Parker, J.; Perou, C. M.; Hammond, S. M. Nat. Methods 2004, 1, 47−53. (20) Liang, R. Q.; Li, W.; Li, Y.; Tan, C. Y.; Li, J. X.; Jin, Y. X.; Ruan, K. C. Nucleic Acids Res. 2005, 33, e17. (21) Beuvink, I.; Kolb, F. A.; Budach, W.; Garnier, A.; Lange, J.; Natt, F.; Dengler, U.; Hall, J.; Filipowicz, W.; Weiler, J. Nucleic Acids Res. 2007, 35, e52. (22) Lee, J. M.; Jung, Y. Angew. Chem., Int. Ed. 2011, 50, 12487− 12490. (23) Chen, C.; Ridzon, D. A.; Broomer, A. J.; Zhou, Z.; 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, e179. (24) Raymond, C. K.; Roberts, B. S.; Garrett-Engele, P.; Lim, L. P.; Johnson, J. M. RNA 2005, 11, 1737−1744. (25) Wan, G.; Lim, Q. E.; Too, H. P. RNA 2010, 16, 1436−1445. (26) Zhang, J.; Li, Z.; Wang, H.; Wang, Y.; Jia, H.; Yan, J. Chem. Commun. 2011, 47, 9465−9467. (27) Tu, Y.; Wu, P.; Zhang, H.; Cai, C. Chem. Commun. 2012, 48, 10718−10720. (28) Xu, F.; Dong, C.; Xie, C.; Ren, J. Chem.Eur. J. 2010, 1010− 1016. (29) Wang, Y.; Zheng, D.; Tan, Q.; Wang, M. X.; Gu, L. Q. Nat. Nanotechnol. 2011, 6, 668−674. (30) Dong, H.; Lei, J.; Ju, H.; Zhi, F.; Wang, H.; Guo, W.; Zhu, Z.; Yan, F. Angew. Chem., Int. Ed. 2012, 51, 4607−4612. (31) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80, 2319−2325. (32) Allawi, H. T.; Dahlberg, J. E.; Olson, S.; Lund, E.; Olson, M.; Ma, W. P.; Takova, T.; Neri, B. P.; Lyamichev, V. I. RNA 2004, 10, 1153−1161. (33) Hartig, J. S.; Grune, I.; Najafi-Shoushtari, S. H.; Famulok, M. J. Am. Chem. Soc. 2004, 126, 722−723. (34) Hackenberg, M.; Sturm, M.; Langenberger, D.; Falcon-Perez, J. M.; Aransay, A. M. Nucleic Acids Res. 2009, 37, w68−76. (35) Jia, H.; Li, Z.; Liu, C.; Cheng, Y. Angew. Chem., Int. Ed. 2010, 49, 5498−5501. (36) Bi, S.; Zhang, J.; Hao, S.; Ding, C.; Zhang, S. Anal. Chem. 2011, 83, 3696−3702. (37) Wang, G. L.; Zhang, C. Y. Anal. Chem. 2012, 84, 7037−7042. (38) Jonstrup, S. P.; Koch, J.; Kjems, J. RNA 2006, 12, 1747−1752. 7947
dx.doi.org/10.1021/ac401715k | Anal. Chem. 2013, 85, 7941−7947
