Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR

Abstract

Here, a method using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER) for miRNAs quantification was established. The strategy has two steps: (1) ligation of two DNA probes specifically hybridize to target miRNA and (2) qPCR amplifying the ligated probe. The miRNA-binding regions of the probes are stem-looped, a motif significantly reduces nonspecific ligation at high ligation temperature (65°C). The ends of the probes are designed complementary to form a paired probe, facilitating the recognition of target miRNAs with low concentrations. RNase H proved to be able to stabilize the heteroduplex formed by the probe and target miRNA, contributing to enhanced sensitivity (limit of detection = 60 copies). High specificity (discriminating homology miRNAs differing only one nucleotide), wide dynamic range (seven orders of magnitude) and ability to accurately detect plant miRNAs (immune to hindrance of 2'-O-methyl moiety) enable SPLICER comparable with the commercially available TaqMan and miRCURY assays. SYBR green I, rather than expensive hydrolysis or locked nucleic acid probes indispensable to TaqMan and miRCURY assays, is adequate for SPLICER. The method was efficient (<1 h), economical ($7 per sample), and robust (able to detect xeno-miRNAs in mammalian bodies), making it a powerful tool for molecular diagnosis and corresponding therapy.

Keywords: MT: Oligonucleotides: Diagnostics and Biosensors; RNase H; SplintR ligase; ligation; miRNA quantification; molecular diagnosis; qPCR.

Graphical abstract

Figure 1

Scheme of SPLICER-qPCR. Probe1 and probe2 are designed regional complementary, so a paired probe could be prepared by annealing the two probes The target miRNA hybridizes with the paired probe to form a heteroduplex with a nick, which is then ligated by SplintR ligase with the aid of RNase H. SMBs are applied in certain application scenarios to remove the background molecules including the non-target miRNAs. The enriched ligated probes proceed to qPCR reactions to realize the quantification of the target miRNA.

Figure 2

Quantitative detection of miRNA159 by the ligation-based method (A) Schematic binding process of miR159 and two specific probes. (B) Sequence of miR159 and its variants. The mutated nucleotides from miR159 were marked in red. “m” indicates 2′-O-methyl modification. (C) Specificity of the miR159-splinted ligation at different temperatures. Ligation reactions were performed using probe 1 and probe 2 that exclusively target miR159 sequence. Urea denaturing PAGE (top) and qPCR (bottom) were employed to analyze the ligation products. “Lig-P,” “Pb-1,” and “Pb-2” denote ligated probe, probe 1, and probe 2. The ligation system contains 2 μL miRNA (5 μM), 3 μL the probes (5 μM each), 1 μL yeast RNA (1 μg/μL), 1 μL 10× SplintR buffer, and 2.5 μL RNase-free water. The solution was incubated at 95°C for 5 min and slowly cooled down to the ligation temperature, followed by the addition of SplintR ligase (0.5 μL) and incubated for 5 min at the designated temperatures. Results of three independent reactions were summarized and shown in the bar charts. (D) Sensitivities of the ligation-based method using the paired (left) and the separated probes (right). Probe 1 and probe 2 were annealed beforehand to form the paired probe by complementary sequences. Gradient diluted miR159 solutions (6 × 10⁷–6 × 10 copies) added with yeast RNA served as the templated for quantification, and each sample was tested in triplicate. The ligation system was as described in (C), and the reactions were performed at 65°C for 5 min, followed by ligase inactivation and qPCR as described in the Materials and methods section.

Figure 3

Addition of RNase H improves the sensitivity and the specificity of SPLICER-qPCR (A) Dosage effect of Rnase H on the ligation products. Rnase H varying in amount (1.2–0.012 U) was introduced to the ligation system. The products were analyzed by urea denaturing PAGE (top), and their relative amounts (bottom) were determined by comparing with that of ligation without Rnase H (0 U). “Lig-P,” “Pb-1,” and “Pb-2” denote ligated probe, probe 1 and probe 2. The ligation system was firstly set up including: 2 μL miRNA (5 μM), 3 μL paired probes (5 μM each), 1 μL yeast RNA (1 μg/μL), and 1 μL 10× SplintR buffer. The mixture was heated at 95°C for 5 min and cooled down to 65°C, before the addition of 2.5 μL RNase H (varying in amount) and 0.5 μL SplintR ligase. The ligation was for 5 min, and terminated by incubation at 95°C for 5 min. (B) Sensitivity of SPLICER-qPCR for quantitatively detecting miR159 in the presence of RNase H. RNase H of 0.02 U was included in ligation of miR159 varying in concentration. Each sample was tested in triplicate, and the mean values were used to plot the standard curve. (C) Effects of RNase H on the specificity of SPLICER-qPCR. The paired probe targeting miR159 was used in ligation reactions for miR159 and its variants (mutated nucleotides marked in red). For ligations with RNase H, 0.02 U was applied. The amount ligation products were determined by qPCR. Results of three independent reactions were summarized and shown in the bar charts. “∗∗” represents an extremely significant difference with p < 0.01.

Figure 4

Detection of the plant-derived miRNAs using SPLICER, miRCURY and TaqMan (A) Quantitative detection of miR156, miR159, and miR168a from broccoli. The total RNA was extracted from fresh broccoli, and aliquots from the same solution were analyzed by the three methods. Results of three independent reactions were summarized and shown in the bar charts. (B) Sensitivities of the three methods to detect the plant-derived miR159 from the diluted RNA extracts. The total RNA extracts subjected to 10 times gradient dilution were used as templates to quantify miR159. Each sample was tested in triplicate. (C) Specific detection of the plant miR159 accompanied with high concentration of miR159-e. Probes and primers of three methods were specific to miR159. miR159-e, which has one mutated nucleotide from miR159, was used as a highly similar sequence to evaluate detection specificity. Each sample was tested in triplicate. Sequence information: miR159 (5′-UUUGGAUUGAAGGGAGCUCUA-3′), miR159-e (5′-UUUGGAUUGCAGGGAGCUCUA-3′), the underlined letter indicates the mutated nucleotide.

Figure 5

Analysis of miRNAs from different mouse tissues using SPLICER, miRCURY, and TaqMan (A) Relative expression of miR-122, miR-24 and Let-7a in mouse tissues as quantified by the three methods. The fold-change of a miRNA determined by one method was calculated based on the C_q values, using the tissue that has the lowest level of the miRNA as base level. Each sample was tested in triplicate, and the mean values were used for plotting. (B) Correlations between the quantitative results from the three methods. Pearson correlation coefficient (R) was used to evaluate the relevance between any two methods. The linear regression curves based on the bivariate distribution were expressed as the gray dotted lines. (C) Correlations between quantification results of Let-7a from different mouse tissues. The total correlation coefficients of Let-7a were expressed by gray circles varying in hue. Red, white, and blue circles indicate positive, weak, and negative correlations, respectively.

Figure 6

Quantitative detection of exogenous miR159 in rat circulation by SPLICER, miRCURY, and TaqMan (A) Time course amplification curves of miR159 in rat peripheral blood introduced by tail vein injection. (B) Dynamic ranges and standard curves of miR159 from the three methods. Data points considered as outliers from dynamic range were marked in gray. (C) Levels of exogenous miR159 in rat serum at 0.5, 1, 2, and 4 h after the tail vein injection. Data were obtained based on the C_q values and standard curves presented in (B). ND, not detected. Each sample was tested in triplicate, and the mean values and the standard deviations were used for plotting the bar charts. Operation of SPLICER was as follows. The ligation system: 2 μL template miRNA, 3 μL mixed probe 1, probe 2 (5 μM each), 1 μL yeast RNA (1 μg/μL), and 1 μL 10× SplintR buffer. The mixture was heated at 95°C for 5 min and slowly cooled down to 65°C, followed by adding RNase H (2.5 μL) and SplintR ligase (0.5 μL), and incubation at 65°C for 5 min. The ligation reaction was terminated at 95°C for 5 min before the qPCR reaction as detailed in the Materials and methods section.