In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

We present here a proof of principle investigation of a novel rolling circle technology for the detection of non-polyadenylated RNA molecules in situ, including a new probe format (the Turtle Probe) and optimized procedures for its use on formalin fixed paraffin embedded tissue sections and in solid support format applications.The method presented combines the high discriminatory power of short oligonucleotide probes with the impressive amplification power and selectivity of the rolling circle reaction, providing excellent signal to noise ratios in combination with exact target localization due to the target primed reaction. Furthermore, the procedure is easily multiplexed, allowing visualization of several different RNAs.DNA and RNA molecules in situ have traditionally been studied by in situ hybridization (ISH). ISH originally utilized probes in the form of radioactively labeled rRNA, visualized by autoradiography [1,2]. Subsequently, various non-isotopic probe labels have also been used, usually detected with immunoenzymatic methods [3] or fluorescent in situ hybridization (FISH) [4,5]. In order to generate sufficient signal, non-isotopic ISH methods usually use long probes or multiple probe cocktails for binding of sufficient number of label molecules to each target. These probes or probe cocktails are in most cases combined with some form of signal amplification such as tyramide signal amplification (TSA), a technique that can increase FISH signal intensity 10–20 fold [6]. However, long probes pose a problem since affinity and specificity for nucleic acid probes usually are inversely correlated, meaning that whilst a probe's affinity for a target increases so does the risk of non-specific binding [7]. Long probes are also not well suited for the discrimination of minor sequence variations. Artificial nucleic acids, such as PNA- and LNA-oligonucleotides, have been utilized as probes, allowing higher hybridization temperatures and increased specificity of the ISH