AbstractThe authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients. Graphical abstractSchematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.

中文翻译：

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基于链置换扩增和滚环扩增的microRNA荧光测定

摘要作者描述了 microRNA 的荧光测定。它基于两步扩增，包括 (a) 链置换复制和 (b) 滚环扩增。链置换扩增系统利用模板 DNA（包含与 microRNA-21 互补的序列）和切口酶位点。杂交后，microRNA 链通过 DNA 聚合酶链反应延伸，然后被切口酶切割。由此产生的 DNA 在滚环扩增中充当引物。然后，添加 DNA 探针 SYBR Green II 以与 ssDNA 结合以产生荧光信号，该信号随着 microRNA 浓度的增加而增加。该方法检测范围广，覆盖10 f。至 0.1 nM microRNA 浓度范围，检测限低至 1.0 fM。该方法成功应用于健康和乳腺癌患者血清中microRNA-21的测定。基于涉及链置换复制和滚环扩增的两步扩增的荧光 microRNA 检测示意图。然后将 DNA 探针 SYBR Green II 与 ssDNA 结合以产生荧光信号，该信号随着 microRNA 浓度的增加而增加。