Padlock-Probes and Rolling-Circle Amplification

Abstract

The padlock-probes and rolling-circle amplification technology is a simple, sensitive, and reliable miRNA expression detection protocol. A padlock-probe is a linear DNA probe where the two terminal aims are designed to be exactly antisense to the 5’-end and 3’-end sequences of a target miRNA of interest. Rolling circle amplification allows linear amplification of the target sequence in a highly quantitative manner without relying on thermocyclers or other advanced read-out systems. The technology was initially developed by Nilsson et al. (Science 265:2085–2088, 1994); Department of Molecular Biology, University of Aarhus, Denmark for detecting localized DNA, and was later modified for detection and distinction of RNA by the same group (Hum Mutat 19:410–415, 2000; Hum Mutat 19:410–415, 2002). Recently, Jonstrup et al. (RNA 12:1747–1752, 2006) further developed the methods into a unique technology for detecting, amplifying, and quantifying miRNA expression. The technology is a simple and reliable miRNA detection protocol and can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA. The techniques have been validated for six miRNAs, namely miR-16, miR-17-5p, miR-20a, miR-21, miR-27a, and miR-92.