The detection of different nucleic acid sequences has diverse applications in molecular biology, mRNA expression studies, genetic analysis, molecular diagnostics and forensic science. In the past decade, microRNA as a potential source for biomarkers had already been applied in monitoring the disease, embryo's pre-natal diagnosis, tumor research. Recently we have developed some novel methods for quantitative detection of miRNAs and demonstrated their practical application in biological samples. One-step real-time PCR assay taking advantages of the enhancement of base-stacking hybridization avoids the reverse transcription step or ligation step to produce the longer template, which commonly is necessary in conventional PCR methods for miRNA analysis. The assay can quantitatively measure the sequence-specific miRNAs in a dynamic range from 500 fM to 10 nM within 40 min with excellent differentiation ability for discrimination of one-base difference among the miRNA family. We have also developed a universal method to quantify miRNA expression levels by target-assisted isothermal exponential amplification coupled with fluorescent DNA-scaffolded AgNCs. The method demonstrates superior ultrasensitivity with a detection limit of miRNA of 2.38 aM (approximate 15 copies of a miRNA molecule in a volume of 10 �L). Taking the advantages of the interesting feature of the fine-tuned emission spectrum of fluorescent DNA/AgNCs, simultaneous detection of multiple miRNAs can be easily realized. The experimental results demonstrated that these simple and cost-effective methods have a potential of becoming the major tool for simultaneous ultrasensitive quantitative analysis of multiple miRNAs (biomarkers) and DNA in tissures or cells for clinical early diagnosis.

Development and application of novel methods for quantitative detection of miRNAs in biological samples

247th National Meeting (2014)

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