Highly Sensitive and Specific Multiplexed MicroRNA Quantification

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Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction (LCR) Pengbo Zhang, Jiangyan Zhang, Chengli Wang, Chenghui Liu, Hui Wang, and Zhengping Li Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Dec 2013 Downloaded from http://pubs.acs.org on December 25, 2013

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Analytical Chemistry

Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction (LCR) Pengbo Zhang,† Jiangyan Zhang,† Chengli Wang,† Chenghui Liu,*,†, ‡ Hui Wang,‡ Zhengping Li*,†,‡ †

Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education;

College of Chemistry and Environmental Science, Hebei University, Baoding 071002, Hebei Province, P. R. China. ‡

Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key

Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, Shaanxi Province, P. R. China Corresponding author: [email protected] (C. Liu); [email protected] (Z. Li). Fax: +86 29 81530859.

ACS Paragon Plus Environment

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ABSTRACT As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. Firstly, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a and mir-143, which can also be simultaneously detected in as small as 2 ng total RNA sample.


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INTRODUCTION MicroRNAs (miRNAs), a class of endogenous and small non-coding RNAs, play an important role in various cellular processes, such as differentiation, cell growth and apoptosis.1 A large number of reports have indicated the role of miRNAs in connection with various diseases such as diabetes, neurological disorders, and especially human cancers.2 Many studies have revealed that miRNA-expression alterations are involved in the initiation and progression of human cancers.3,4 Cancer-specific miRNA fingerprints have been identified in various types of human cancers,4,5 including breast cancer, ovarian cancer, lung cancer, gastric carcinoma, and colon carcinoma.6-8 These findings highlight the potential of these miRNA fingerprints in cancer diagnosis, progression, prognosis and response to treatment.3,4 These miRNA fingerprints generally involve the expression alterations of several miRNA species. Therefore, sensitive, specific and multiplexed miRNA detection has great significance for cancer diagnosis and treatment as well as for the studies of the tumorigenesis. Many sensitive assays for miRNA detection have recently been developed based on enzymatic amplification techniques, such as polymerase chain reaction (PCR),9-11 modified invader assay,12 rolling circle amplification,13,14 exponentially isothermal amplification,15-19 in which high sensitivity and specificity can be achieved with the enzymatic amplification reactions in homogeneous solution. However, these assays are ill-suited for multiplexed miRNA detection. To date, microarray-based assays

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and massively sequencing methods

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are most widely used

for multiplexed miRNA detection due to their high-throughput screening capability. However, the reagents and the equipments of these methods are too expensive, which make them only suitable to be used in large laboratories where thousands of samples are detected daily, but not


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suitable for clinical diagnosis. 22 Moreover, several unique characteristics of miRNAs, including their short length, sequence similarity and wide ranges of natural abundance make the sensitivity and specificity of microarray-based methods difficult to satisfy the needs of multiplexed miRNA quantification.23 There are several significant strategies developed to improve the sensitivity for miRNA detection with surface-based approaches by using new labeled probes, including Au nanoparticles,24-26

quantum

dot,27,28

silver

nanoclusters,29

OsO2

nanoparticles,30

and

bioluminescent probe.31 Although these surface-based approaches may be implemented directly on a microarray surface, these techniques are still in their early stages and perhaps require significant cares to make them practical for microarrays.32 Several studies have shown that miRNA fingerprints generally require 2~15 miRNA species to distinguish cancerous from noncancerous tissues.

6, 32

Therefore, simple and cost-effective

methods for multiplexed detection of several miRNA species are now desired for clinical diagnosis. The instrument of capillary electrophoresis (CE) is available in ordinary laboratories and cost-effective for detection of several miRNA species. Krylov and coworkers have explored the direct quantitative analysis of multiple miRNAs (DQAMmiR) based on hybridization between tagged DNA probes and miRNA targets and CE separation.32,33 DQAMmiR methods can detect multiple miRNAs without the requirement of miRNA modification. However, the direct hybridization-based approaches are limited in their sensitivity (with detection limit of 100 pM) and the ability to discriminate highly related miRNAs. By using the size-coded ligationmediated PCR, Faridani et al. have greatly improved the sensitivity (with detection limit of 1.0 pM) with PCR amplification and the specificity based on the prerequisite of a ligation reaction.34 Doyle et al. have further pushed the detection limit of multiplexed miRNA quantification down to subfemtomolar by using rolling circle amplification (RCA) on encoded gel microparticles.35


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However, the RCA-based method needs a time-consuming procedure, generally taking 10 h for completion. With the thermal cycles of ligation reaction, ligase chain reaction (LCR) can obtain high specificity to discriminate one-base mutation in DNA targets and high sensitivity comparable to PCR for genomic DNA detection.36, 37 In a previous report,38 we have demonstrated that short miRNAs are suitable to be detected by LCR. Unfortunately, although high specificity can be achieved, the sensitivity (with detection limit of 0.7 pM) of the LCR-based miRNA assay is not satisfactory. In this work, we modify one of the DNA probes with two ribonucleotides, which can greatly improve the efficiency and specificity for ligation reaction of DNA probes templated by RNAs.39 With the modified DNA probe, the proposed LCR-based assay can detect miRNAs as low as 0.2 fM, increasing the sensitivity with 3 orders of magnitude. Moreover, by coding the DNA probes with different length of oligo (dA), different miRNAs can produce LCR products with different length, which can be simply separated with CE to realize multiplexed miRNA detection. EXPERIMENTAL SECTION Materials and Reagents. T4 RNA ligase 2, T4 RNA ligase reaction buffer, Adenosine 5'Triphosphate (ATP) and Taq DNA ligase were purchased from New England Biolabs. HPLCpurified miRNAs, ribonucleotide-modified DNA probes, dNTPs, Ribonuclease inhibitor and DEPC-treated water were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). PAGE-purified DNA oligonucleotides were obtained from Shanghai Sangon Biotech. (Shanghai, China). The sequences of all RNA and DNA used were available in Table S-1 (see Supporting Information). Salmon sperm DNA solution was purchased from Invitrogen (Beijing, China). All


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the solutions for ligation reactions were prepared in DEPC-treated water. All other reagents were of analytical reagent grade and used as purchased without further purification. Total RNA Extraction. Human lung adenocarcinoma epithelial cell line A549, human leukemia cell line K562, and human renal epithelial cell line 293T were cultured in RPMI 1640 (GIBCO) while human breast cancer cell line MDA-MB-453 and MDA-MB-231 were cultured in L15 (HyClone) and DMEM (GIBCO), respectively. Total RNA samples were isolated from these cell lines by using Trizol® Reagent (Invitrogen, Beijing, China) following the manufacturer’s protocol. The concentration of total RNA was determined from the absorption at 260 nm with TU-1901 UV-VIS Spectrophotometer (PGeneral, Beijing, China). Ligation of DNA Probes Templated by MiRNA. The ligation reaction mixture consists of 2 nM probe A-M, 2 nM probe B, 50 µM ATP, 20 U Ribonuclease inhibitor and ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT). After addition of appropriate amount of target miRNA or total RNA samples, the mixture was heated at 65ºC for 2 min and 39ºC for 3 min. Then 1 U T4 RNA ligase 2 was added in the mixture to a final volume of 10 µL, and the reaction mixture was incubated at 39ºC for 35 min to complete the ligation reaction. After the ligation reaction, the products were immediately put on ice. LCR Amplification Reaction. 2 µL of the ligation product was transferred to 10 µL LCR reaction mixture containing 2 µg salmon sperm DNA, probe A, probe B, probe A' and probe B' (each concentration was 10 nM). The reaction mixture was heated at 95ºC for 1 min and 80ºC for 2 min. Then 8 µL mixture containing 4 U Taq DNA ligase and the reaction buffer (20 mM TrisHCl, pH 7.6, 25 mM KOAc, 10 mM Mg(OAc)2, 10 mM DTT, 1 mM NAD, 0.1% Triton® X100) was added in the reaction mixture at 80ºC. The LCR reaction was carried out with a 2720


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thermocycler (Applied Biosystems, USA) at following 35 thermal cycles: 95ºC for 30 sec and 65ºC for 2 min. CE separation and Data Analysis. CE separation was performed using ABI Prism 310 Genetic Analyzer (Applied Biosystems, USA). The LCR products were diluted 10-fold with water, and then 1 µL dilution was mixed with 18.8 µL of Hi-Di formamide (Applied Biosystems) and 0.2 µL of GeneScan 120 LIZ Size Standard (Applied Biosystems). Then the samples were denatured at 95ºC for 5 min and cooled on ice for 10 min. 47 cm × 50 µm capillary and POP-4 polymer purchased from Applied Biosystems were used for CE separation of LCR products. The parameters for each run were: injection time 10 sec, injection and run voltage 10 kV, temperature 60ºC, and run time 24 min. Experimental data were analyzed using the GeneMapper 4.1 software (Applied Biosystems) and the positions and areas of the peaks were determined. RESULTS AND DISCUSSION Principle of the LCR-based Multiplexed MiRNA Detection. Usually, T4 DNA ligase is used to ligate DNA probes templated by DNA and RNA.40 T4 RNA ligase 2 can efficiently join nicks in the RNA strand of a RNA:RNA or DNA:RNA hybrid. It has been demonstrated that T4 RNA ligase 2 has higher specificity for ligation of DNA probes templated by RNA than that of T4 DNA ligase.13 Moreover, a DNA probe modified with two ribonucleotides (defined as probe A-M) can greatly improve the efficiency of the ligation reaction of DNA probes by using miRNAs as the templates.39 Therefore, T4 RNA ligase 2 and probe A-M are adopted in the initial ligation step. The principle is schematically described in Figure 1. Briefly, probe A-M and probe B (modified with a phosphate group at its 5'-terminus) are hybridized to a target miRNA. To simplify the probe design, the miRNA target-specific sequences in probe A-M and probe B are


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designed to cover half of the miRNA sequence in order to keep similar Tm between probe A-M and probe B. Subsequently, T4 RNA ligase 2 joins the adjacently hybridized probes to form the template AMB. Then, probe A' (fluorescence-labeled at its 3'-terminus and modified with a phosphate group at its 5'-terminus) and probe B' are hybridized to AMB and ligated with thermostable Taq DNA ligase to produce another template A'B'. The probe A, probe B and probe A', probe B' are respectively hybridized to A'B' and AMB at 65ºC and ligated to form the templates of AB and A'B' again. After denaturation by heating to 95ºC, AMB, AB and A'B' can act as the templates for next step of ligation reactions. The sequence of probe A is the same as that of probe A-M but without modification with ribonucleotides at its 3'-terminus. Accordingly, through the thermal cycles of 95ºC for 30 sec and 65ºC for 2 min, the ligation products from one round can become the templates to ligate the DNA probes for next round of ligation to form the ligase chain reaction (LCR) which gives rise to exponential amplification and finally produces a large amount of double stranded AB/A'B'. The probe B' is designed to have a different length tag of oligo (dA) as a ‘size code’ for different target miRNAs. Therefore, the ligation products of A'B' bear a fluorescent label (from probe A') and have different length for different target miRNAs. When the LCR products (AB/A'B') are separated by CE under denaturing conditions and detected with laser induced fluorescence (LIF), different target miRNAs can simultaneously be detected in one LCR reaction.


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Figure 1. Schematic representation of the LCR-based multiplexed miRNA assay. The Advantage of Probe A-M Modified with Two Ribonucleotides. We first test whether the probe A-M modified with two ribonucleotides at its 3′-terminus can improve the ligation efficiency of DNA probes templated by miRNA. By using let-7a as the miRNA target, the ligation reaction in the first step shown in Figure 1 is performed with let-7a probe A-M, let-7a probe B and let-7a probe A and let-7a probe B, respectively. Then, the LCR amplification, CE separation and LIF detection are performed with the same procedures as described in the experimental section. As depicted in Figure 2, by using probe A without modification in the ligation reaction, the signals produced by let-7a in the concentration range from 0.2 fM to 20 fM cannot obviously be distinguished from the blank. Let-7a can be detected only when its


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concentration is greater than 200 fM. However, by using probe A-M modified with two ribonucleotides at its 3'-terminus in the ligation step, as low as 0.2 fM let-7a can give the signal three-fold greater than the blank. The results prove that the modification of DNA probes with ribonucleotides can greatly improve the ligation efficiency, and sequentially, increase the sensitivity for miRNA detection with 3 orders of magnitude.

Figure 2. The relative signals produced by let-7a miRNA at different concentrations in the LCRbased assay respectively using probe A and probe A-M in the ligation step. The calculation of ligation productivity (%) is described below in the section of Dynamic Range and Sensitivity. [Probe A] or [probe A-M] = 2 nM, [probe B] = 2 nM, [T4 RNA ligase 2] = 1 Unit. The blank is detected in the same procedure as the let-7a detection but without let-7a miRNA. Optimization of the Amounts of Salmon Sperm DNA in LCR Reaction. The non-specific amplification is always an inevitable problem for the exponential amplification of nucleic acids, which is the main cause to limit the sensitivity for the detection of nucleic acid targets. Addition of salmon sperm DNA in the LCR reaction can block the generation of non-specific amplification.36 Therefore, the optimization of the amounts of salmon sperm DNA is investigated. Let-7a miRNA at different concentrations is respectively detected with the


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proposed assay according to the procedures described in experimental section except the amounts of salmon sperm DNA. As shown in Figure 3, when 0.5 µg salmon sperm DNA is used in the LCR reaction, the blank signal is large, indicating the seriously non-specific amplification. The signal produced by 0.2 fM let-7a is only 1.35 fold of the blank signal. When 2 µg salmon sperm DNA is added to the LCR reaction, the blank signal is greatly reduced and the signal ratio of 0.2 fM let-7a to the blank reaches 3.5. When the amount of salmon sperm DNA is increased to 4 µg in the LCR reaction, the blank signal can be negligible. However, the signal produced by 0.2 fM let-7a is also not detectable. Obviously, salmon sperm DNA can efficiently reduce the non-specific amplification in LCR reaction, and unavoidably, also reduce the target-specific signal. As described above, 2 µg salmon sperm DNA is found to be optimum for the LCR-based assay, which produces small blank signal and maximum signal ratio of miRNA target to blank.

Figure 3. Effect of the amount of salmon sperm DNA on the LCR-based miRNA assay. The signals are obtained according to the procedure described in the experimental section except that 0.5, 2, 4 µg salmon sperm DNA is added in the LCR reaction, respectively. Optimization of Temperature and Time for Ligation Reaction. As we know, the high specificity of miRNA assay for discriminating one-base differences between miRNAs is very


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important due to the high similarity among miRNA sequences. In our LCR-based miRNA assay, the temperature and reaction time of the ligation reaction play important roles for achieving high selectivity. Therefore, the optimization of the temperature and time for the ligation reaction has been investigated.

Figure 4. (a) Effect of the temperature of ligation reaction on miRNA detection. The relative signal produced by let-7a at the ligation temperature of 35°C is defined as 100%. The concentrations of let-7a and let-7c are both 20 fM; (b) Effect of the ligation reaction time on the detection of let-7a. The concentration of let-7a is 1 fM. Other experimental conditions are the same as described in the Experimental Section. Error bars are estimated from the standard deviation of three repetitive measurements. The effect of ligation temperature on miRNA detection is studied by using let-7a and let-7c as the model targets because they are different only by one-base and they are most difficult to be discriminated among the let-7 miRNA family members. The ligation reaction by using let-7aspecific probes, is performed at 35°C, 37°C, 39°C and 41°C, respectively, templated by let-7a and let-7c. After ligation and LCR reaction, the LCR products are separated and detected by CE. As shown in Figure 4a, when the ligation temperature is increased from 35°C to 39°C, the signal of let-7a shows a slow decrease but the signal of let-7c decreases sharply. When the ligation temperature is further increased from 39°C to 41°C, the signal of let-7a decreases remarkably but


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the interference of let-7c can not be obviously improved. So it can be seen that the signal ratio of let-7a to let-7c reached the highest value at 39°C. Therefore, taking into account of both specificity and sensitivity for miRNA detection, 39°C is selected as the ligation temperature in this study. Under the selected ligation temperature of 39°C, the ligation time is subsequently investigated. As shown in Figure 4b, the ligation productivity (LP%) produced by let-7a show rapid increase at the beginning but slow increase when the ligation time is longer than 20 min. On the other hand, the blank signal will obviously increase when ligation time is greater than 35 min. The signal ratio of let-7a to the blank reached the highest value at 35 min. Therefore, 35 min is selected as the ligation time in this work. Dynamic Range and Sensitivity. The dynamic range and sensitivity of the LCR-based assay for miRNA quantification are evaluated to detect serial dilutions of a synthetic let-7a miRNA target. As shown in Figure S-1 (Supporting Information), as low as 0.2 fM let-7a can be accurately determined. As increasing the concentration of let-7a, the peak height and peak area of LCR products are increased, while those of fluorescence-labeled probe A' are correspondingly decreased. In order to minish analysis bias resulted from different run of CE-LIF detection, the ligation productivity (LP %) is used for the quantitative detection of the miRNAs, which can be calculated as follows: Ligation productivity (LP%) =

peak area of LCR product × 100% peak area of LCR product + peak area of probe A'

As demonstrated in Figure 5, there is a good linear relationship between LP (%) and the logarithm (log) of let-7a concentration in the range of 0.2 ~ 100 fM. The correlation equation is LP (%) =489.5 + 30.1 lgCmiRNA(M) and the corresponding correlation coefficient R is 0.9993,


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indicating that the LCR-based assay is quantitative and has a widely linear range over four orders of magnitude.

Figure 5. The relationship between ligation productivity (%) and log of let-7a concentration (M). The concentration of let-7a is 0, 0.2, 1, 2, 10, 20, 100 fM, respectively. Error bars are estimated from the standard deviation of three repetitive measurements. Specificity Study. Due to the sequence homology of miRNAs, especially for miRNA family members, the specificity of miRNA assay to distinguish very similar sequences of miRNA targets is very important. The let-7 miRNA family members (let-7a~g and let-7i) consist of highly similar eight miRNAs that differ by only 1~4 nucleotide from each other (Figure 6), which provides a challenging set for the specificity of detection methods. Therefore, the let-7 miRNA family members are chosen as the model to test specificity of the LCR-based miRNA assay. For the ligation reaction, the specificity is generally very high when the ligation junction is located at the site of single-nucleotide mismatch. However, for miRNA targets, it is not possible to design the probes in which the ligation junction is just located at the site of distinct nucleotides because of the short length of miRNA sequences. Therefore, to investigate the specificity of the proposed LCR-based miRNA assay, the let-7 miRNA family members are


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detected by using let-7a probe A-M and let-7a probe B (see the sequences in Table S-1), in which the specific sequence is respectively complementary to half of the let-7a sequence. One can see from Figure 6 that let-7a can be clearly distinguished against all other let-7 miRNA family members. It is worth noting that there is only a single different nucleotide between let-7a and let-7c, let-7a and let-7e, let-7a and let-7f. Compared to let-7a, the mismatched nucleotide in let-7f just locates at the ligation site (the 3'-terminus of probe A-M). Therefore, the nonspecific detection of let-7f is very little (0.05%). For let-7e, the different nucleotide compared to let-7a is near to the ligation site (with three nucleotide space). So let-7e produces larger nonspecific detection (11.9%) than that of let-7f. The mismatched nucleotide in let-7c is near the 3'-terminus of the miRNA sequence, and thus, far away from the ligation site, therefore, let-7c produces maximum nonspecific detection (17.9%). Although let-7b has two different nucleotides compared to let-7a, the different nucleotides are both near the 3'-terminus of the miRNA sequence. So let-7b also produces 6.07% nonspecific detection. Except described above, let-7d, let-7g and let-7i have 2-4 nucleotides differed from let-7a. All these miRNAs produce very little nonspecific detection (0.17%, 0.01% and 0.06%, respectively). Collectively, the LCR-based assay can be characterized with high specificity to discriminate a single nucleotide difference among miRNA targets.


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Figure 6. The specificity evaluation for the LCR-based assay. The relative detection of let-7a is normalized to 100% and the relative detection of other miRNAs is calculated by the correlation equation from Figure 5. The concentration of each let-7 miRNAs is 20 fM. The different nucleotides compared to let-7a in other let-7 miRNA family members are underlined. Multiplexed MiRNA Detection. To test the multiplexing capability for miRNA detection of the LCR-based miRNA assay, let-7a, mir-92a and mir-143 are randomly chosen as the model miRNA targets. The probe A-M, probe B, probe A' and probe B' designed for detection of these miRNA targets are listed in Table S-1 (Supporting Information). As described formerly, the probe B' has a different length of oligo (dA) tag for different miRNA targets (the oligo(dA) length is 2, 6, and 18 respectively for let-7a, mir-92a and mir-143). Modulated by the probe B', the fluorescence-labeled A'B' in the LCR products is 66 nucleotide for let-7a, 70 nucleotide for mir-92a and 82 nucleotide for mir-143, respectively. These three miRNAs are firstly detected with the LCR-based assay, individually. Then the miRNAs are mixed together and simultaneously detected with one ligation and LCR reaction. As shown in Figure 7 a ~ e, the LCR products (A'B') derived from different miRNAs can be well separated and detected. The size of the LCR product of each miRNA is identical to that obtained from the individual assay. In addition, the effects of the length of oligo(dA) on ligation efficiency have been studied through detection of let-7a by using probe B' with different length of oligo(dA). As shown in Figure S-2, the ligation productivity (LP %) for detection of 50 fM let-7a is calculated to be 86.56%, 85.78% and 83.29% when the oligo(dA) length is 2, 6, and 18 respectively. The results indicate that the ligation efficiency gradually decreases with increasing the length of oligo(dA). However, the influence of the length of oligo(dA) on ligation efficiency is very little. Moreover,


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the length of oligo(dA) is fixed for detection of one kind of miRNA. Therefore, effects of the length of oligo(dA) on miRNA detection should be negligible. The interferences of other let-7 family members on the detection of let-7a are also investigated in the multiplexed miRNA detection system. As can be seen from the results shown in Figure S-3 and Figure S-4, the interferences of other let-7 miRNA family members in the multiplexed miRNAs detection system are similar to those in detection of single let-7a target (shown in Figure 6), further demonstrating the good specificity of the proposed method for multiplexed miRNA detection. In a final set of experiments, the LCR-based assay is employed to detect multiple miRNA targets in a total RNA sample extracted from A549 cell line. As demonstrated in Figure 7f, welldefined signals for let-7a and mir-92a can be observed, but the signal of mir-143 is not detectable. In order to test whether the LCR-based assay can detect mir-143 in the sample, we add 20 fM mir-143 into the total RNA sample and detect it with the same experimental procedure. The result shown in Figure 7g proves that mir-143 can be well detected in the sample, indicating that mir-143 expression should be below the detectable level of the LCR-based assay. To further testify the practicality of the proposed LCR method for multiplexed miRNA detection, a series of total RNA samples extracted from four types of cell lines are prepared and the contents of let-7a, mir-92a and mir-143 in these total RNA samples are determined respectively by the proposed LCR method and commercial Stem-loop RT-PCR Kits by using TaqMan probes (ordered from Life Technology, Applied Biosystems, USA). The comparison of the determination results for these samples by using the commercial RT-PCR kits and the proposed LCR method is shown in Table S-2. It can be seen that the relative errors of the proposed LCR method compared to the commercial RT-PCR method are less than ±20% with


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two exceptions, further demonstrating that the proposed LCR approach is practical for multiplexed miRNA quantification in real samples.

Figure 7. The electropherograms for detection of miRNAs and total RNA samples. (a) blank, (b) 20 fM let-7a, (c) 20 fM mir-92a, (d) 20 fM mir-143, (e) 20 fM let-7a, 20 fM mir-92a and 20 fM mir-143, (f) 2 ng total RNA sample, (g) 2 ng total RNA sample spiked with 20 fM mi-143. Blank is detected with the same procedure described in the experiment section mentioned above but without miRNA target, while for b ~ d contains only one miRNA target. The abscissa axis in the


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electropherogram represents the retention time and the longitudinal axis represents the relative fluorescence signal. CONCLUSIONS Here we have demonstrated that the design of DNA probe modified with two ribonucleotides at its 3'-terminus can greatly increase the ligation efficiency of DNA probes by using miRNAs as the templates, and thus, dramatically improve the sensitivity of the LCR-based assay for miRNA detection. With the modified DNA probes, as low as 0.2 fM miRNA targets can be accurately detected and a single nucleotide difference among miRNA sequences can be discriminated with high specificity. By simply encoding the DNA probes with different length of oligo (dA) for different miRNA targets, the LCR-based assay can be employed to detect multiple miRNA targets in one ligation and LCR reaction with a commercial CE-LIF instrument. All experimental procedures of the LCR-based multiplexed miRNA detection only need about 3h. In our proof of principle work, three miRNAs can be simultaneously detected in as low as 2 ng total RNA sample. Similar to the size-coded LCR, the multiplex ligation-dependent probe amplification (MLPA) technique is based on size-coded ligation mediated PCR amplification and CE separation.41 The MLPA method can detect up to 50 different DNA sequences in a single reaction. So the ligation reaction and CE separation should possess the potential multiplexing ability to detect miRNAs of up to 50 different species. As described above, miRNA fingerprints for clinical diagnosis generally require 2~15 miRNA species.6, 32 Therefore, the proposed LCRbased assay may provide a valuable tool to detect multiple miRNA signature for research and diagnostics applications. ASSOCIATED CONTENT


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Supporting

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Information. The sequences of miRNAs and the DNA probes, the

electropherograms for detection of let-7a at different concentrations, effects of the length of oligo(dA) on the ligation efficiency, interferences of other let-7 family members on the detection of let-7a in the multiplexed miRNA detection system and the comparison of the proposed LCR method and RT-PCR kits for the detection of miRNAs from different cell lines. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * [email protected] (C. Liu); * [email protected] (Z. Li) ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (20925519, 91127035, 21335005), Program for Changjiang Scholars and Innovative Research Team in University (IRT 1124), Doctoral Fund of Ministry of Education of China (20111301130001).


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REFERENCES (1) Cheng, A. M.; Byrom, M. W.; Shelton, J.; Ford, L. P. Nucleic Acids Res. 2005, 33, 1290– 1297. (2) Arenz, C. Angew. Chem. Int. Ed. 2006, 45, 5048–5050. (3) Calin, G. A.; Croce, C. M. Nat. Rev. Cancer 2006, 6, 857–866. (4) 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. (5) 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. (6) Iorio, M. V.; Ferracin, M.; Liu, C. G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; Ménard, S.; Palazzo, J. P.; Rosenberg, A.; Musiani, P.; Volinia, S.; Nenci, I.; Calin, G. A.; Querzoli, P.; Negrini, M.; Croce, C. M. Cancer Res. 2005, 65, 7065–7070. (7) Iorio, M. V.; Visone, R.; Leva, G. D.; Donati, V.; Petrocca, F.; Casalini, P.; Taccioli, C.; Volinia, S.; Liu, C. G.; Alder, H.; Calin, G. A.; Ménard, S.; Croce, C. M. Cancer Res., 2007, 67, 8699–8707.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

(8) Calin, G. A.; Croce, C. M. Cancer Res., 2006, 66, 7390–7394. (9) 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. (10) Raymond, C. K.; Roberts, B. S.; Garrett-Engele, P.; Lim, L. P.; Johnson, J. M. RNA 2005, 11, 1737–1744. (11) Li, J.; Yao, B.; Huang, H.; Wang, Z.; Sun, C.; Fan, Y.; Chang, Q.; Li, S.; Wang, X.; Xi, J. Anal. Chem. 2009, 81, 5446–5451. (12) 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. (13) Cheng, Y.; Zhang, X.; Li, Z.; Jiao, X.; Wang, Y.; Zhang, Y. Angew. Chem. Int. Ed. 2009, 48, 3268–3272. (14) Wen, Y.; Xu, Y.; Mao, X.; Wei, Y.; Song, H.; Chen, N.; Huang, Q.; Fan, C.; Li, D. Anal. Chem. 2012, 84, 7664–7669. (15) Jia, H.; Li, Z.; Liu, C.; Cheng, Y. Angew. Chem. Int. Ed. 2010, 49, 5498–5501. (16) Duan, R.; Zuo, X.; Wang, S.; Quan, X.; Chen, D.; Chen, Z.; Jiang, L.; Fan, C.; Xia, F. J. Am. Chem. Soc. 2013, 135, 4604–4607. (17) Zhang, Y.; Zhang, C. Y. Anal. Chem. 2012, 84, 224–231. (18) Liu, Y. Q.; Zhang, M.; Yin, B. C.; Ye, B. C. Anal. Chem. 2012, 84, 5165–5169.


22

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19) Bi, S.; Zhang, J.; Hao, S.; Ding, C.; Zhang, S. Anal. Chem. 2011, 83, 3696–3702. (20) Thomson, J. M.; Parker, J.; Perou, C. M.; Hammond, S. M. Nat. Methods 2004, 1, 47–53. (21) Morin, R. D.; O'Connor, M. D.; Griffith, M.; Kuchenbauer, F.; Delaney, A.; Prabhu, A.-L.; Zhao, Y.; McDonald, H.; Zeng, T.; Hirst, M.; Eaves, C. J.; Marra, M. A. Genome Res. 2008, 18, 610–621. (22) Cissell, K. A.; Shrestha, S.; Deo, S. K. Anal. Chem. 2007, 79, 4754–4761. (23) Wark, A. W.; Lee, H. J.; Corn, R. M. Angew. Chem. Int. Ed. 2008, 47, 644–652. (24) Fang, S.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044– 14046. (25) Johnson, B. N.; Mutharasan, R. Anal. Chem. 2012, 84, 10426–10436. (26) Wang, J.; Yi, X.; Tang, H.; Han, H.; Wu, M.; Zhou, F. Anal. Chem. 2012, 84, 6400–6406. (27) 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. (28) Li, J.; Schachermeyer, S.; Wang, Y.; Yin, Y.; Zhong, W. Anal. Chem. 2009, 81, 9723– 9729. (29) Dong, H.; Jin, S.; Ju, H.; Hao, K.; Xu, L. P.; Lu, H.; Zhang, X. Anal. Chem. 2012, 84, 8670–8674. (30) Gao, Z.; Yang, Z. Anal. Chem. 2006, 78, 1470–1477. (31) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80,


23


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Page 24 of 25

2319–2325. (32) Wegman, D. W.; Cherney, L. T.; Yousef, G. M.; Krylov, S. N. Anal. Chem. 2013, 85, 6518–6523. (33) Wegman, D. W.; Krylov, S. N. Angew. Chem. Int. Ed. 2011, 50, 10335–10339. (34) Arefian, E.; Kiani, J.; Soleimani, M.; Shariati, S. A. M.; Aghaee-Bakhtiari, S. H.; Atashi, A.; Gheisari, Y.; Ahmadbeigi, N.; Banaei-Moghaddam, A. M.; Naderi, M.; Namvarasl, N.; Good, L.; Faridani, O. R. Nucleic Acids Res., 2011, 39, e80. (35) Chapin, S. C.; Doyle, P. S. Anal. Chem. 2011, 83, 7179–7185. (36) Barany, F. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 189–193. (37) Cheng, Y.; Du, Q.; Wang, L.; Jia, H.; Li, Z. Anal. Chem. 2012, 84, 3739–3744. (38) Yan, J.; Li, Z.; Liu, C.; Cheng, Y. Chem. Commun. 2010, 46, 2432–2434. (39) Nandakumar, J.; Shuman, S. Mol. Cell 2004, 16, 211–221. (40) Nilsson, M.; Barbany, G.; Antson, D. O.; Gertow, K.; Landegren, U. Nat. Biotechnol. 2000, 18, 791–793. (41) Sørensen, K. M.; Andersen, P. S.; Larsen, L.A.; Schwartz, M.; Schouten, J. P.; Nygren, A. O. H. Anal. Chem. 2008, 80, 9363–9368.


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for TOC only


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Highly Sensitive and Specific Multiplexed MicroRNA Quantification

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