Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation

Schneider, Nils; Meier, Matthias

doi:10.1261/rna.057836.116

Cited by 25 publications

(15 citation statements)

References 29 publications

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“…As reported earlier, we also observed a high rate of non-cellular amplicons with SplintR ligase 19 | P a g e concentrations above 1 µM 22 . RCA mix consisting of 0.6 U/µl of Phi29 Polymerase (Lucigen, 30221-2) in 1X Phi29 polymerase buffer, 0.25 mM dNTPs, in DEPC treated water was added immediately after ligation step.…”

Section: High-content Boloramis Assaysupporting

confidence: 86%

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

Iyer

Punthambaker

Liu

et al. 2018

Preprint

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We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallelIn-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotides in length and upwards, from over 200, 000 individual human induced pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-tocell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications. Manuscript:Single-cell transcriptomics is an exponentially evolving field, with recent developments in multiplexed in-situ technologies paving the way for spatial imaging of the genome and transcriptome at an unprecedented resolution 1 . We proposed Fluorescent In Situ Sequencing (FISSEQ) in 2003, and in 2014 demonstrated the generation and sequencing of highly multiplexed, spatially resolved in-situ RNA libraries in cells and tissues 2,3 . While FISSEQ's novelty lay in enabling unbiased discovery, it is primarily limited by poor detection sensitivity, and unsuitability for targeted in-situ transcriptomics (<0.005% compared to single-molecule (sm) FISH) 4,5 . Padlock probes have been demonstrated for in-situ sequencing for multiplexed transcriptomics, with singlebase resolution on a small number of transcripts 6 . However in both methods, detection efficiency is a function of RT efficiency, and subject to noise resulting from variable priming efficiency and 3 | P a g e random priming induced bias 7 . LNA modified primers have been demonstrated to increase RT efficiency with padlock probes (~30%), but require careful calibration and can be costprohibitively expensive for genome-wide applications (~2 order higher cost than unmodified primers) 5,6,8 . smFISH based multiplexed transcriptome imaging methods overcome the limitations of RT by directly hybridizing a plurality of oligo-paint like encoded DNA probes directly on target RNA, and subsequently reading out target locations using a two-stage hybridization scheme 9-14 . While smFISH based methods offer the highest in-situ RNA detection efficiency, it is best suited for large transcripts (>1500nt) and can yield lower signal than RCA based methods (~50-200 vs ~800-1000 fluorophores/transcript, respectively). As a result, a vast segment of biologically interesting RNA species, (including short-non coding RNA) remain vastly inaccessible to Multiplexed In situ (MIS) methods 1,15 . Consequently, there is a strong demand for robust MIS RNA detection methods that can overcome some of the limitations of each method (sensitivity, cost barrier and transcript-size limitation), while retaining the desirable features of these ...

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Section: High-content Boloramis Assaysupporting

confidence: 86%

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

Iyer

Punthambaker

Liu

et al. 2018

Preprint

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“…Two recent papers 35 , 36 reported that in situ RCA can detect eukaryotic mRNA using a padlock probe and SplintR ligase. Previously, RCA for detecting eukaryotic mRNA was performed after RT using target-specific primers 34 .…”

Section: Discussionmentioning

confidence: 99%

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

Takahashi

Ohkawachi

Horio

et al. 2018

Sci Rep

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RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3′-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

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“…This enzyme is a DNA ligase encoded by Paramecium bursaria Chlorella virus 1, first discovered by Ho et al,(29). A number of studies have successfully applied SplintR ligase for RNA species detection in cultured cells (26, 30–32). However, the fidelity of SplintR ligation in end-joining is questionable because it tolerates mismatches at the padlock probe junction and its usefulness is debated.…”

Section: Introductionmentioning

confidence: 99%

SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

Sountoulidis¹,

Liontos²,

Nguyen³

et al. 2020

Preprint

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16 17 ǂ Corresponding authors: Christos Samakovlis (christos.samakovlis@scilifelab.se), Alexandros 18 Sountoulidis (alexandros.sountoulidis@scilifelab.se) 19 20 Keywords: SCRINSHOT, padlock-probe, multiplex RNA FISH, spatial cell-type map, lung 21 22 2 23 Abstract 24 Changes in cell identities and positions underlie tissue development and disease progression. 25 Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of 26 cell-states, spatially resolved single-cell mapping presents a challenging task. We developed 27 SCRINSHOT (Single Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex 28 RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by 29 circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock 30 probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles 31 thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on 32 murine and human organs. SCRINSHOT quantification of marker gene expression shows high 33 correlation with published scRNA-Seq data over a broad range of gene expression levels. We 34 demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types 35 along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT 36 facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and 37 experimental conditions. 38 39 40 41 42 43 44 45 65addressed by the sequential fluorescence in situ hybridization (seqFISH) (14, 15) and the 66 multiplexed error-robust FISH (MERFISH) (4). These approaches utilize sequential rounds of 67 hybridization of FISH probes or barcode-based primary probes to detect multiple RNA species. 68The outstanding throughput of these methods makes them strong candidates for generation of 69 spatial transcriptome maps in tissues. However, since the principle of these techniques is similar 70 to smFISH, they require large number of gene-specific probes, confocal or super-resolution 4 71 microscopy to deconvolve the signals and complicated algorithms for both probe design and 72 analysis. Nevertheless, the low signal-to-noise ratio still remains a major technical challenge of 73 these methods, especially for tissue sections with strong auto-fluorescence from structural 74 extracellular matrix components like collagen and elastin (16). New strategies for signal 75 amplifications, such as branched-DNA amplification (RNAScope) (17) and hybridization chain 76 reaction (18, 19), have been recently combined with sophisticated probe design (AmpFISH) (20) 77 to increase sensitivity and specificity of smFISH. 78 Padlock probes have been successfully used to detect RNA species (21). They are linear DNA 79 molecules, with complementary arms to the target mRNA sequence and a common "backbone". 80 Upon hybridization with the target sequence, they can be ligated, creating circular single-stranded 81...

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Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation

Cited by 25 publications

References 29 publications

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

Barcoded oligonucleotides ligated on RNA amplified for multiplex and parallel in-situ analyses

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

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