title: Highly sensitive and specific multiplexed microRNA quantification using size-coded ligation chain reaction.
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. First, 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 of total RNA sample.
authors.affiliation: 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.
authors.forename: Pengbo
authors.initials: P
authors.lastname: Zhang
authors.forename: Jiangyan
authors.initials: J
authors.lastname: Zhang
authors.forename: Chengli
authors.initials: C
authors.lastname: Wang
authors.forename: Chenghui
authors.initials: C
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Li
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D045744:Cell Line, Tumor; D011088:DNA Ligases / Q000737:chemistry; D015342:DNA Probes / Q000138:chemical synthesis; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide
chemical_list: D014408:Biomarkers, Tumor; D015342:DNA Probes; C542359:MIRN143 microRNA, human; C541874:MIRN92 microRNA, human; D035683:MicroRNAs; C487931:mirnlet7 microRNA, human; D011088:DNA Ligases