In situdetection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS
© Stougaard et al; licensee BioMed Central Ltd. 2007
Received: 27 April 2007
Accepted: 18 October 2007
Published: 18 October 2007
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.
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  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 . 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 . 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-probes [8, 9]. Short horseradish peroxidase (HRP) conjugated oligonucleotides have been used for detection of RNA, using TSA and fluorescently labeled antibodies . Traditional ISH-methods often rely strictly on hybridization and stringent washing for specificity. Therefore, background staining will increase along with the specific signals as result of non-specific binding of the probe. This limits detection of rare targets . Furthermore 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 .
Another method used for in situ detection of nucleic acids is the primed in situ labeling (PRINS) technique. The PRINS technique is based on the generation of detectable DNA by performing a DNA polymerization in situ. Traditionally this has been done by using short synthetic oligonucleotides which are hybridized to a target nucleic acid, and used as primers in the subsequent DNA polymerization step during which hapten- or fluorescent-labeled nucleotides are incorporated for tagging sites of DNA synthesis . PRINS has usually been performed on repetitive DNA sequences [13, 14], although it has been shown to allow both single copy gene detection , and mRNA detection .
Thus, existing in situ detection technologies rely on target nucleic acids being sufficiently large or abundant to be detected, and minor molecular differences in individual molecules may be beyond the limits of detection. We now present an approach for RNA detection combining the best from PRINS and FISH. Initially, an "inversed" PRINS reaction is performed in which a hybridization probe (that can be ligated to form a closed circle) is used as template and the target nucleic acid acts as primer (the opposite of the traditional PRINS approach). The subsequent DNA polymerization results in the tagging of sites of DNA synthesis with tandem repeat copies of the circular probe. This firmly localized repeated sequence can then easily be detected by FISH. The whole reaction can readily be multiplexed through the application of a cocktail of probes and subsequent visualization of the individual PRINS products with color coded identifier oligonucleotides.
Such an approach was recently presented for the analysis of DNA molecules in situ , combining Padlock Probes for DNA detection at single nucleotide resolution, with target primed rolling circle DNA synthesis, resulting in an amplification powerful enough to allow single molecule detection. ISH with circle probes and target primed rolling circle requires both hybridization of the probe and the presence of a 3'-end to prime the rolling circle reaction and thus is unlikely to give any signals if the probe attaches non-specifically to the target specimen. Furthermore, only part of the circle probe hybridizes to the target and by using different sequences in the non-binding part the individual probes can easily be identified in multiplex assays.
We now report a new design according to this concept, in which circular hybridization probes detect non-polyadenylated RNA molecules, initiating a rolling circle PRINS reaction from the natural 3'-end of the target RNA molecule hybridized to the probe. For this, we developed a new type of hybridization probe, a "Turtle Probe", and made modifications to previous protocols.
Results and discussion
Optimization and generation of new probe format
In parallel with the optimization of the reaction procedures, we therefore supplemented the Padlock format with circle probes not requiring target templated ligation. One way of obtaining such probes would be by using preformed circles. Preformed circles were known before Padlock Probes, and have been employed for in situ detection of DNA . However, the performance of the probes in that in situ study was not impressive compared to the performance of Padlock Probes in situ , and since the circles are formed on external linear templates, there is always the risk that some template molecules are carried over into the in situ reaction, where they could initiate rolling circle replication of probes not hybridized to the proper target. Since the amplification from the rolling circle reaction is sufficient for single molecule detection, any template molecule carried over would be a potential source of false signals. In practice, perfect removal of template molecules is time and labor consuming – if at all possible – since it requires e.g. gelbased purification and it would, therefore, be preferable to leave out this template. To this end we designed what we call Turtle Probes (Figure 1B). These have the desirable features of templating their own formation into closed circles and of only generating signals upon hybridization to and priming from the hybridization target. For in situ detection Turtle Probes have the added potential advantage over preformed circles of being closed by ligation after target hybridization, which may make them better able to wrap around the target RNA with its potential modifications and cross-links to other bio-molecules in the preparation.
While target primed detection has its advantage in securing target localization, it is also limited by the need for a properly located 3'-end in the target molecule. A collaborative study with the laboratory of Ulf Landegren on how far from the 3'-end a Padlock Probe could be positioned on a DNA template in situ in ethanol fixed cell lines and still result in signal showed that while signals could still be obtained with a probe positioned 134 nucleotides from the 3'-end, this position gave less signals than a position closer to the end of the target molecule . Although similar tests have not been performed on FFPE tissue with RNA as target instead of DNA, the effect of the distance from the 3'-end will most likely be even more marked here. In the case of RNA, the matter is further complicated by the tendency of RNA to form secondary structures, and we have unpublished experimental indications on pure RNA in solution that the recession of the 3'-end stops when the first region of double stranded RNA is reached.
In situdetection with Turtle Probes
Next we tested the two types of probes on FFPE tissue material. Here we initially chose the Epstein-Barr Virus (EBV) encoded RNA, EBER1 (Epstein-Barr early RNA1), as target, since EBER1, besides being non-polyadenylated and present in high amounts in EBV positive tumors such as Hodgkin's lymphoma, also provides an internal negative control with EBER1 primarily located in the nuclei of the neoplastic Reed-Sternberg cells in this setting . As a second target, we selected the RNA template for human telomerase (hTR) since, in addition to being non-polyadenylated and much less abundant than EBER1, it is also expected to be found in a subset of cells (i.e. in all Reed-Sternberg cells and less abundantly in some of the non-malignant lymphoid bystander cells) . Furthermore, hTR should give rise to a few discrete signals in the nuclei, reflecting accumulation in Cajal bodies [17, 24].
The present paper represents to our knowledge the first publication of RNA detection in situ with oligonucleotide probes and target primed rolling circle PRINS. This approach appears promising because of the high discriminatory power of the short oligonucleotide probes and the impressive amplification power 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 may be multiplexed to a significant extent, color-coding a number of probes reacted in parallel for combinatorial labeling. As illustrated, the approach works also when challenged with the most technically difficult preparation format, that of FFPE routine samples from the pathology archives, opening up these collections to sensitive and specific RNA analysis, something that has been possible only to a limited extent. Additionally, the technology should be appealing for the analysis of the rapidly expanding universe of small structural and regulatory RNA molecules (e.g. si- and miRNAs) [25, 26]. Furthermore, we have just recently, while this paper was under revision, published a method using similar self-templating probes in combination with rolling circle DNA synthesis for production of long 5'-phosphorylated oligonucleotides .
The in vitro transcribed HPV16E6 RNA was made by purifying genomic DNA (gDNA) from SiHa cells using QIAamp DNA mini kit (Qiagen). The gDNA, either from yeast cells (donated by Torben H. Jensen) or from SiHa cells, was used as template in a PCR, using the Expand High Fidelity PCR System (Roche), and primers for either HPV16E6 or SSA4. The sequences of the primers were, for SiHa gDNA, senseHPV16-E6-T7promoter (GGAAGAAGCT TAATACGACT CACTATAGGG ATGCACCAAA AGAGAACTGC AAT) and antisenseHPV16-E6 (AGGGAATTCG AATGCGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTACAG CTGGGTTTCT CTACGTG), and for yeast gDNA, senseSSA4-3'UTR-T7promoter (GGAAGAAGCT TAATACGACT CACTATAGGG ATAAATACAA AGATGC) and antisense3'SSA-3'UTR (AGGGAATTCA ATTAACCCTC ACTAAAGGGT CGTGTTGTTT GGCG). Both sense primers contained the minimal promoter sequence recognized by the T7 RNA polymerase and a HindIII recognition site. The antisense primer for SiHa gDNA contained a stretch of 30 thymine nucleotides (to provide the RNA transcript with an artificial polyA tail), as well as both an EcoRI and a BsmI recognition site, whereas the antisense primer for yeast gDNA only had an EcoRI recognition site. The PCR products were purified by gel electrophoresis using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences now GE Healthcare) and inserted into the pUC18 plasmid (Fermentas) using the restriction enzymes HindIII and EcoRI, to provide the plasmids named pUC18-E6a and pUC18-SSA4-3'UTR. Both plasmids (amplified in competent XL1-blue bacteria) were digested with either BsmI (for generation of the HPV16E6a RNA), AflIII (for generation of the HPV16E6-noPA RNA), or EcoRI (for generation of the SSA4-3'UTR RNA fragment), purified by phenol-chloroform extraction and precipitated with ethanol prior to transcription. The in vitro transcription was performed with the T7 transcription kit (Fermentas) using the restriction digested plasmids as transcription template, and the RNA was purified by polyacrylamide gel electrophoresis (PAGE).
Detection of RNA on solid support
Linking and hybridization reactions
The capture oligonucleotides, 3'AmHPV16E6 (GTCATATACC TCACGTCGCA GTAACTGTTG CCTTCCTTCC TTCCTT -Amin-3') and 3'AmSSA4 (AGGGAAAACT AAGAAATTCG ATGCTGCTAC CCTTCCTTCC TTCCTT-Amin-3'), were coupled to CodeLink activated slides (Amersham Bioscience) according to the manufactures protocol, except that the spotted area (approximately 2.5 mm2) was encircled with a DAKO-pen (Dako) and spotted by adding 10 μL of capture oligonucleotide mixture to the slide. The following reactions were all performed in a total reaction volume of 5 μL. Following all hybridizations or enzymatic reactions, the slides were washed in wash buffer (0.1 M Tris-HCL (pH 7.5, at 25°C), 0.15 M NaCl, 0.05% Tween-20) at room temperature (RT). The duration of the wash was 3 min after the hybridization steps and 1 min after the enzymatic steps. In vitro transcribed RNA (0.25 μM RNA was used in a total volume of 5 μL hybridization mixture, corresponding to final amounts of approximately 21 ng SSA4 RNA, 37 ng HPV16E6-noPA and 41 ng HPV16E6a, respectively) was hybridized to the capture oligonucleotide in a mixture containing: 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5, at 25°C), 1 mM EDTA, 0.01% SDS, 10 mM DTT and 1 u/μL Ribolock RNase Inhibitor (Fermentas). The hybridization reaction took place in a humidity chamber over night (16 hours) at 37°C. The Turtle Probes, TP-SSA4end-id16 (p-GTCGATCCCC TCAATGCTGC TGCTGTACTA CAATTCAATT AACCCTCACT AAAGGGTCGT GGGATCGACT CGGAATAACC GA) and TP-PolyA-id33 (p-CATTCTCCCC TCAATGCACA TGTTTGGCTC CTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TGGAGAATGC GAGAATAACT CG) were hybridized in a final concentration of 0.1 μM in a total volume of 5 μL, under the same conditions as the RNA, though only for 30 min at 37°C in the humidity chamber.
Ligation was performed with 0.1 u/μL T4 DNA Ligase (Fermentas) in 1× T4 DNA ligation buffer (supplied with the T4 DNA ligase) supplemented with 1 u/μL Ribolock RNase Inhibitor (Fermentas) and 0.2 μg/μL BSA in a humidity chamber for 30 min at 37°C. The rolling circle was performed with 1 u/μL phi29 DNA polymerase (Fermentas) in 1× phi29 buffer (supplied with the phi29 DNA polymerase) supplemented with 1 u/μL Ribolock RNase Inhibitor (Fermentas), 0.2 μg/μL BSA and 0.25 mM dNTP in a humidity chamber for 30 min at 37°C.
Hybridization of detection probes
The fluorescent probes, Lin16 (5'-Rhodamine-CCTCAATGCT GCTGCTGTAC TAC) and Lin33 (5'-FITC-CCTCAATGCA CATGTTTGGC TCC) were hybridized to the rolling circle product in a concentration of 0.2 μM of each in a total volume of 5 μL, in a mixture containing 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5, at 25°C), 1 mM EDTA, 0.01% SDS, 10 mM DTT and 1 u/μL Ribolock RNase Inhibitor (Fermentas) in a humidity chamber for 30 min at 37°C. The slides were washed in wash buffer 2 × 3 min, dehydrated in 99% EtOH, drained for excess ethanol, air dried, and mounted with VectaShield (Vector Laboratories).
Detection of RNA in FFPE cell line
Pretreatment of FFPE cell line
Hela cells were grown to confluenc, spun down and the cell pellet was fixed in 10% buffered formalin and embedded in paraffin. Sections (4 μm thick) were cut from the FFPE Hela cell pellet block using a standard microtome, placed on SuperFrost®Plus glass slides (Menzler-gläser) and incubated for 45 min at 65°C. The sections were deparaffinized in xylene for 2 × 10 min. The xylene was extracted in 99.9% (vol/vol) ethanol for 4 × 2 min, in 85% (vol/vol) ethanol for 2 × 2 min, and in 99.9% (vol/vol) ethanol for 1 × 2 min, the slides were drained for excess ethanol and air dried. The sections were submerged in 0.6 u/μL pepsin (solid units, Sigma) in 0.1 M HCl and incubated at 37°C for 8 min. Pepsin concentration and incubation time may need to be optimized according to the length of fixation, the type of tissue, and the type of sample. Pepsin treatment was stopped by submerging slides in wash buffer for 2 min (wash buffer: 0.1 M Tris-HCL (pH 7.5, at 25°C), 0.15 M NaCl, 0.05% Tween-20), after which the slides were dehydrated through an ethanol series (70%, 85%, 99.9% (vol/vol)), drained for excess ethanol, and air dried.
The tissue sections were encircled with a DAKO-pen (Dako), and a hybridization mixture containing 20% formamide, 2× SSC, 5% glycerol, 1 μg/μL carrier RNA (Qiagen) and 100 nM, 10 nM or 1 nM Turtle Probe, TP-5S rRNA (p-GTCGATCCCC TCAATGCACA TGTTTGGCTC CAAAGCCTAC AGCACCCGGT ATTCCCAGGC GGGATCGACT CGGAATAACC GA), TP-28S rRNA (p-GTCGATCCCC TCAATGCACA TGTTTGGCTC CGACAAACCC TTGTGTCGAG GGCTGACTTT CGGATCGACT CGGAATAACC GA), TP-hTR (p-GTCGATCCCC TCAATGCTGC TGCTGTACTA CGCATGTGTG AGCCGAGTCC TGGGTGCACG TCCCACAGCT CGGATCGACT CGGAATAACC GA), or Padlock Probe, PP-5S rRNA (p-CGGTATTCCC AGGCGTTTAT TTCCTCAATG CACATGTTTG GCTCCTAGTG ATTTACTTGG ATGTCTAAAG CCTACAGCAC C), PP-28S rRNA (p-CGAGGGCTGA CTTTCTTTAT TTCCTCAATG CACATGTTTG GCTCCTAGTG ATTTACTTGG ATGTCTGACA AACCCTTGTG T), PP-hTR (p-TGGGTGCACG TCCCACAGCT CTTTATTTCC TCAATGCTGC TGCTGTACTA CTAGTGATTT ACTTGGATGT CTGCATGTGT GAGCCGAGTC C) was added to the slide. The hybridization was performed under a coverslip sealed with heat-resistant glue, and for optimal temperature control the slide hybridization was performed using a Twin Tower PTC-200 (MJ Research, Waltham, MA, USA) programmed as follows: Heat to 95°C for 2 min, RAMP 0.5°C/s to 37°C and 37°C for 30 min. The slides may be left at 37°C as long as overnight, provided they do not dry out during the extended incubation. To remove unbound probe, slides were washed separately for 5 min in 2× SSC + 0.05% Tween-20 preheated to 37°C, and washed for 5 min in wash buffer preheated to 37°C. (Slides were washed separately since single molecule sensitivity carries the inherent risk of cross contamination).
Ligation of Turtle Probes was performed in 1× T4 DNA ligation buffer (supplied with the T4 DNA ligase), 0.2 μg/μL BSA, and 0.1u/μL T4 DNA ligase (Fermentas). Ligation of Padlock Probes was performed in the buffer described in Nilsson et al. (2000). Ligation reactions, were incubated under coverslip at 37°C for 30 min in a humidity chamber, washed in wash buffer for 3 min at room temperature, dehydrated and air-dried. The rolling circle was performed in 1× Phi29 buffer (supplied with the Phi29 DNA polymerase), 0.2 μg/μL BSA, 0.25 mM dNTP, 5% glycerol, and 1 u/μL Phi29 DNA polymerase (Fermentas) under a coverslip at 37°C for 45 min in a humidity chamber. Following the rolling circle, slides were washed in wash buffer for 3 min at RT, dehydrated and air-dried.
Hybridization of detection probes
The rolling circle products were detected by hybridizing the fluorescently labeled identifier probes lin16 and lin33 (0.2 μM of each) in a mixture containing 20% formamide, 2× SSC, and 5% glycerol under coverslip for 30 min at 37°C in a humidity chamber (may be left over night at 37°C). The slides were washed in wash buffer 2 × 5 min at room temperature, dehydrated, and mounted with VectaShield with DAPI (Vector Laboratories).
Detection of RNA in FFPE tissue
All steps were performed as for detection of RNA in FFPE cell line except that the Turtle Probe, TP-hTR (p-GTCGATCCCC TCAATGCTGC TGCTGTACTA CGCATGTGTG AGCCGAGTCC TGGGTGCACG TCCCACAGCT CGGATCGACT CGGAATAACC GA), TP-EBER1 (p-GTCGATCCCC TCAATGCACA TGTTTGGCTC CAAAACATGC GGACCACCAG CTGGTACTTG ACCGGATCGA CTCGGAATAA CCGA) or Padlock Probe, PP-EBER1 (p-CAGCTGGTAC TTGACCCCTC AATGCTGCTG CTGTACTACT AGTGATTTAC TTAAAACATG CGGACCAC) was used in a final concentration of 100 nM.
Both solid support- and tissue-slides were analyzed in a Leica epifluorescence microscope and images recorded with a SenSys CCD-camera operated by the SmartCapture 2 version 2.0 image analysis from digitalscientific (Cambridge, UK).
This work was supported by grants from the EU (Framework 6, MolTools consortium), the John and Birthe Meyer Foundation, the Aase og Ejnar Danielsens Foundation, the Civilingeniør Frode V. Nyegarrd og Hustrus Foundation and the Arvid Nilssons Foundation. We thank Associate Professors Anni Andersen and Birgitta Knudsen from the Institute of Molecular Biology at Aarhus University for providing the competent cells, Associate Professor Simon Turk and Post Doc. Morten Krog Larsen from the Laboratory of Developmental Genetics at Umeå University for assistance with handling and culturing of C. elegans, Associate Professor Torben Heick Jensen from the Institute of Molecular Biology at Aarhus University for the genomic yeast DNA, PhD-student Brita Singers from the Department of Oncology at Aarhus University Hospital for providing the Siha cells, and Professor Thomas Ledet from the Laboratorie for Biochemical Pathology at Aarhus University Hospital for providing cell culture facilities.
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