Sensitive and Specific Detection of MicroRNAs by Northern Blot Analysis Using LNA-modified Oligonucleotide Probes

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2004 Dec 16

PMID 15598818

Citations 199

Authors

Anna Valoczi

Csaba Hornyik

Nora Varga

Jozsef Burgyan

Sakari Kauppinen

Zoltan Havelda

Affiliations

Soon will be listed here.

Abstract

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.

Citing Articles

Sensitive and accurate photoluminescent-multiphonon resonant Raman scattering dual-mode detection of microRNA-21 via catalytic hairpin assembly amplification and magnetic assistance.

Wen X, Xue Z, Wang K, Li J, Ding Y, Wang G Mikrochim Acta. 2025; 192(1):49.

PMID: 39747697 DOI: 10.1007/s00604-024-06920-1.

Near-infrared DNA biosensors based on polysulfonate coatings for the sensitive detection of microRNAs.

Lin X, Yang Y, Zhu W, He X, Liu Y Nanoscale Adv. 2024; 7(2):549-559.

PMID: 39650618 PMC: 11618855. DOI: 10.1039/d4na00712c.

A Review of Nanotechnology in microRNA Detection and Drug Delivery.

Wang H Cells. 2024; 13(15.

PMID: 39120308 PMC: 11311607. DOI: 10.3390/cells13151277.

Recent progress in Surface-Enhanced Raman Spectroscopy detection of biomarkers in liquid biopsy for breast cancer.

Liu X, Jia Y, Zheng C Front Oncol. 2024; 14:1400498.

PMID: 39040452 PMC: 11260621. DOI: 10.3389/fonc.2024.1400498.

Sensitive fluorescence detection of miRNA-124 in cardiomyocytes under oxidative stress using a nucleic acid probe.

Li S, Pei X, Liu X, Wang S, Xu W, Wang J Heliyon. 2024; 10(13):e33588.

PMID: 39040278 PMC: 11260977. DOI: 10.1016/j.heliyon.2024.e33588.

References

Nielsen K, Rasmussen J, Kumar R, Wengel J, Jacobsen J, Petersen M . NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid. Bioconjug Chem. 2004; 15(3):449-57. DOI: 10.1021/bc034145h. View

Lee R, Feinbaum R, Ambros V . The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993; 75(5):843-54. DOI: 10.1016/0092-8674(93)90529-y. View

Reinhart B, Slack F, Basson M, Pasquinelli A, Bettinger J, Rougvie A . The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000; 403(6772):901-6. DOI: 10.1038/35002607. View

Wahlestedt C, Salmi P, Good L, Kela J, JOHNSSON T, Hokfelt T . Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci U S A. 2000; 97(10):5633-8. PMC: 25880. DOI: 10.1073/pnas.97.10.5633. View

Bondensgaard K, Petersen M, Singh S, Rajwanshi V, Kumar R, Wengel J . Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity. Chemistry. 2000; 6(15):2687-95. DOI: 10.1002/1521-3765(20000804)6:15<2687::aid-chem2687>3.0.co;2-u. View

Braasch D, Corey D . Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem Biol. 2001; 8(1):1-7. DOI: 10.1016/s1074-5521(00)00058-2. View

Rauhut R, Lendeckel W, Tuschl T . Identification of novel genes coding for small expressed RNAs. Science. 2001; 294(5543):853-8. DOI: 10.1126/science.1064921. View

Lee R, Ambros V . An extensive class of small RNAs in Caenorhabditis elegans. Science. 2001; 294(5543):862-4. DOI: 10.1126/science.1065329. View

Ambros V . microRNAs: tiny regulators with great potential. Cell. 2002; 107(7):823-6. DOI: 10.1016/s0092-8674(01)00616-x. View

10.

Jacobsen N, Fenger M, Bentzen J, Rasmussen S, Jakobsen M, Fenstholt J . Genotyping of the apolipoprotein B R3500Q mutation using immobilized locked nucleic acid capture probes. Clin Chem. 2002; 48(4):657-60. View

11.

Kurreck J, Wyszko E, Gillen C, Erdmann V . Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 2002; 30(9):1911-8. PMC: 113840. DOI: 10.1093/nar/30.9.1911. View

12.

Petersen M, Bondensgaard K, Wengel J, Jacobsen J . Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids. J Am Chem Soc. 2002; 124(21):5974-82. DOI: 10.1021/ja012288d. View

13.

Reinhart B, Weinstein E, Rhoades M, Bartel B, Bartel D . MicroRNAs in plants. Genes Dev. 2002; 16(13):1616-26. PMC: 186362. DOI: 10.1101/gad.1004402. View

14.

Simeonov A, Nikiforov T . Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. Nucleic Acids Res. 2002; 30(17):e91. PMC: 137436. DOI: 10.1093/nar/gnf090. View

15.

Hutvagner G, Zamore P . A microRNA in a multiple-turnover RNAi enzyme complex. Science. 2002; 297(5589):2056-60. DOI: 10.1126/science.1073827. View

16.

Jacobsen N, Bentzen J, Meldgaard M, Jakobsen M, Fenger M, Kauppinen S . LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E. Nucleic Acids Res. 2002; 30(19):e100. PMC: 140560. DOI: 10.1093/nar/gnf099. View

17.

Calin G, Dumitru C, Shimizu M, Bichi R, Zupo S, Noch E . Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002; 99(24):15524-9. PMC: 137750. DOI: 10.1073/pnas.242606799. View

18.

Braasch D, Liu Y, Corey D . Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Res. 2002; 30(23):5160-7. PMC: 137965. DOI: 10.1093/nar/gkf651. View

19.

Mouritzen P, Nielsen A, Pfundheller H, Choleva Y, Kongsbak L, Moller S . Single nucleotide polymorphism genotyping using locked nucleic acid (LNA). Expert Rev Mol Diagn. 2003; 3(1):27-38. DOI: 10.1586/14737159.3.1.27. View

20.

Fluiter K, Ten Asbroek A, de Wissel M, Jakobs M, Wissenbach M, Olsson H . In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides. Nucleic Acids Res. 2003; 31(3):953-62. PMC: 149205. DOI: 10.1093/nar/gkg185. View