Quadratic isothermal amplification for the detection of microRNA

Abstract

This protocol describes an isothermal amplification approach for ultrasensitive detection of specific microRNAs (miRNAs). It achieves this level of sensitivity through quadratic amplification of the target oligonucleotide by using a Bst DNA polymerase-induced strand-displacement reaction and a lambda exonuclease-aided recycling reaction. First, the target miRNA binds to a specifically designed molecular beacon, causing it to become a fluorescence emitter. A primer then binds to the activated beacon, and Bst polymerase initiates the synthesis of a double-stranded DNA segment templated on the molecular beacon. This causes the concomitant release of the target miRNA from the beacon--the first round of 'recycling'. Second, the duplex beacon thus produced is a suitable substrate for a nicking enzyme present in solution. After the duplex beacon is nicked, the lambda exonuclease digests the beacon and releases the DNA single strand just synthesized, which is complementary to the molecular beacon, inducing the second round of recycling. The miRNA detection limit of this protocol is 10 fmol at 37 °C and 1 amol at 4 °C. This approach also affords high selectivity when applied to miRNA extracted from MCF-7 and PC3 cell lines and even from breast cancer tissue samples. Upon isolation of miRNA, the detection process can be completed in ∼2 h.

MnO2 Nanotube-Based NanoSearchlight for Imaging of Multiple MicroRNAs in Live Cells

“…1–7 Extensive research efforts have been paid to explore sensitive technologies for the detection of miRNAs. 8–13 Furthermore, it is of great significance to measure a panel of miRNAs and thus give precise and accurate detection of cancers. 14,15 On the other hand, imaging of miRNAs in situ in living cells could recognize cancerous cells directly.…”

Microfluidic System for Detection of Viral RNA in Blood Using a Barcode Fluorescence Reporter and a Photocleavable Capture Probe

“…In addition, PCR requires precise temperature control and expensive and sensitive enzymes. 49,50 Our technique only requires microliters of raw blood and does not need additional sample amplification steps. Using the 10× on-chip concentration protocol, our technique shows a detection limit of 800 aM for Ebola RNA targets, which is comparable with PCR analysis.…”

Multiplex quantitative analysis of microRNA expression via exponential isothermal amplification and conformation-sensitive DNA separation

“…Furthermore, multiplexing power was increased to an eight-plex assay by using high-resolution CE-SSCP as the detection method. Conventional isothermal EXPAR methods are generally able to simultaneously measure the expression levels of only one to three miRNAs 9 – 11 , 13 , 40 . Furthermore, combining isothermal EXPAR with CE-SSCP presents the advantage of having universal amplifiers that can amplify different sets of miRNA targets.…”