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

Magnus Stougaard,Jakob S Lohmann,Stephen Hamilton-Dutoit,Magdalena Zajac,Jørn Koch

doi:10.1186/1472-6750-7-69

Abstract

BackgroundIn situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybridization for specificity can result in non-specific signals appearing anywhere the probe attaches to the target specimen. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis. This has previously been used for DNA detection in situ, but not until now for RNA targets.ResultsWe 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.ConclusionThe 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.

Highlights

In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling
We report a new design according to this concept, in which circular hybridization probes detect non-polyadenylated RNA molecules, initiating a rolling circle primed in situ labeling (PRINS) reaction from the natural 3'-end of the target RNA molecule hybridized to the probe
Padlock Probes [18] are linear probes that are turned into closed circles when the 3'- and 5'-ends are brought into proximity by hybridization to a matching sequence, and the resulting nick is closed by a DNA ligase (Figure 1A)

Summary

Introduction

In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis This has previously been used for DNA detection in situ, but not until now for RNA targets. 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 (page number not for citation purposes). Most amplification techniques used, such as TSA, are not well suited for multiplexing and since both specific and non-specific signals are amplified careful optimization of each hybridization event is required [12]

Methods

Results

Conclusion