Journal
MOLECULAR BIOLOGY REPORTS
Volume 45, Issue 4, Pages 611-619Publisher
SPRINGER
DOI: 10.1007/s11033-018-4200-1
Keywords
MicroRNA (miRNA); Poly(A) tailing assay; Extension-based assay; RT-qPCR; Reverse transcription
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Funding
- Soonchunhyang University
- Ministry of Trade, Industry and Energy of the Republic of Korea [10053770]
- Korea Evaluation Institute of Industrial Technology (KEIT) [10053770] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay consists of poly(A) tailing and reverse transcription followed by real-time PCR. In the first step of this reaction, target miRNAs are poly(A) tailed by poly(A) polymerase followed by cDNA synthesis using poly(T) adaptors. In the second step, cDNA is hybridized to the 3'-end of a specific extension sequence that contains part of a miRNA sequence; this cDNA-specific extension sequence hybrid forms the novel PCR template. The PCR template is amplified in a SYBR Green-based quantitative real-time PCR with universal forward and reverse primers. The miR-106b in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative extension-based assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms.
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