title: Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.
abstract: A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.
authors.affiliation: Institute of Biotechnology, National Taipei University of Technology, 1, Section 3, Taipei 106, Taiwan.
authors.forename: Hsiang-Ping
authors.initials: HP
authors.lastname: Yu
authors.forename: Yi-Ling
authors.initials: YL
authors.lastname: Hsiao
authors.forename: Hung-Yin
authors.initials: HY
authors.lastname: Pan
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Huang
authors.forename: Shao-Yi
authors.initials: SY
authors.lastname: Hou
mesh_terms: D001483:Base Sequence; D001671:Biochemistry / Q000379:methods*; D001681:Biological Assay; D045744:Cell Line, Tumor; D005591:Chemical Fractionation; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D011756:Diphosphates / Q000032:analysis*; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012015:Reference Standards
chemical_list: D011756:Diphosphates; D035683:MicroRNAs; D004259:DNA-Directed DNA Polymerase