Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR
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....
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| Format: | Article |
| Language: | English |
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Elsevier
2023-03-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253122003365 |
| _version_ | 1797955407731228672 |
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| author | Xinshu Qin Xingyu Wang Ke Xu Yi Zhang Hongye Tian Yinglei Li Bangran Qi Xingbin Yang |
| author_facet | Xinshu Qin Xingyu Wang Ke Xu Yi Zhang Hongye Tian Yinglei Li Bangran Qi Xingbin Yang |
| author_sort | Xinshu Qin |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T23:32:38Z |
| format | Article |
| id | doaj.art-3987910751f747899c9dc19c372c8fc9 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-10T23:32:38Z |
| publishDate | 2023-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-3987910751f747899c9dc19c372c8fc92023-01-12T04:18:57ZengElsevierMolecular Therapy: Nucleic Acids2162-25312023-03-0131241255Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCRXinshu Qin0Xingyu Wang1Ke Xu2Yi Zhang3Hongye Tian4Yinglei Li5Bangran Qi6Xingbin Yang7College of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710062, Shaanxi, ChinaCollege of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710062, Shaanxi, China; Corresponding author: Xingyu Wang, College of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710054, Shaanxi, China.Department of Joint Surgery, Hong Hui Hospital, Xi’an Jiaotong University, Xi’an 710054, Shaanxi, ChinaDepartment of Food Science, The Pennsylvania State University, University Park, PA 16802, USACollege of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710062, Shaanxi, ChinaCollege of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710062, Shaanxi, ChinaCollege of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710062, Shaanxi, ChinaCollege of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710062, Shaanxi, China; Corresponding author: Xingbin Yang, College of Food Engineering and Nutritional Science, Shaanxi Normal University, No. 620 West Chang’an Avenue, Xi’an 710054, Shaanxi, China.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.http://www.sciencedirect.com/science/article/pii/S2162253122003365MT: Oligonucleotides: Diagnostics and Biosensorsmolecular diagnosismiRNA quantificationligationqPCRSplintR ligase |
| spellingShingle | Xinshu Qin Xingyu Wang Ke Xu Yi Zhang Hongye Tian Yinglei Li Bangran Qi Xingbin Yang Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR Molecular Therapy: Nucleic Acids MT: Oligonucleotides: Diagnostics and Biosensors molecular diagnosis miRNA quantification ligation qPCR SplintR ligase |
| title | Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR |
| title_full | Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR |
| title_fullStr | Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR |
| title_full_unstemmed | Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR |
| title_short | Quantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR |
| title_sort | quantitative analysis of mirnas using splintr ligase mediated ligation of complementary pairing probes enhanced by rnase h splicer qpcr |
| topic | MT: Oligonucleotides: Diagnostics and Biosensors molecular diagnosis miRNA quantification ligation qPCR SplintR ligase |
| url | http://www.sciencedirect.com/science/article/pii/S2162253122003365 |
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