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


RESULTS
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.


CONCLUSIONS
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.
Stougaard
Magnus
M
Institute of Pathology, Aarhus University Hospital, Nørrebrogade 44 building 18A, DK-8000 Aarhus C, Denmark. mstou@as.aaa.dk
Lohmann
Jakob S
JS
Zajac
Magdalena
M
Hamilton-Dutoit
Stephen
S
Koch
Jørn
J
D005557:Formaldehyde; D006367:HeLa Cells; D006801:Humans; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes / Q000235:genetics*; D016612:Paraffin Embedding; D020401:Primed In Situ Labeling / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000032:analysis / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D016707:Tissue Fixation
D015345:Oligonucleotide Probes; D012339:RNA, Ribosomal, 28S; D012341:RNA, Ribosomal, 5S; D005557:Formaldehyde; D012313:RNA