dc.description.abstract | MicroRNA (miRNA) is a small non-coding RNA molecule, playing an essential role in the expression and regulation of genes. The expression patterns of miRNAs are important to the verification of their predicted function. miRNA levels have been demonstrated to have a strong correlation with disease progression in cancer, which can be considered as diagnostic and prognostic biomarkers. Currently, there are many ways for analyzing miRNA, such as qRT-PCR assays, microarray assays, in situ hybridization(ISH) and the next-generation sequencing(NGS). However, the challenge of detecting specific miRNA and remains
because of its small size, sequence similarity among the miRNA family members. In recent years, many novel nucleotide derivatives have been proposed to gain better signals of sensitivity and specificity such as Locked Nucleic Acid (LNA) and Peptide nucleic acid (PNA).
In this study, we developed DNA containing neutral methyl phosphotriester internucleotide (MPTE) linkages apply in ISH and qPCR.
The use of MPTE modified DNA sequence could reduce electrostatic repulsion effect, and as a consequance, could enhance the duplex formation which can specific and sensitive detection of microRNAs.
In situ hybridization is the only method which can provides insight into both the level and localization in single cell. We used MPTE modified probe for the detection of mimic exogenous miR-524-5p that transfected into HCT116 cell lines (human colon cancer cell lines) and the expression of endogenous miR-21 and miR-29a. We successfully demonstrated improved hybridization efficiency and show the high mismatch discrimination with high signal noise ratio in different temperature.
Quantitative polymerase chain reaction (qPCR) is a powerful method, which has been widely used to quantify the miRNA expression. However, miRNAs in the same family often have overlapping targets or single nucleotide polymorphism(SNP) that are not easy to be discriminated by unmodified DNA primers. we use miR-29a and miR-29c as targets which are both miRNA-29 (miR-29) family share the same mature sequence in vitro, while differ in one nucleotide. Using DNA containing MPTE linkages as primers to enhance the specificity of the SNP discrimination. By adjusting modification number of MPTE modified primer and annealing temperature, we achieve the optimum operating conditions for precise detection of miRNAs.
Depend on the success of applying DNA containing MPTE linkages in ISH and qPCR methods, it could be expected the potential ability of MPTE modified oligonucleotides developing into different biomolecular detection platform and possibly theoretic agent in the future.
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