In Situ Single-Molecule RNA Genotyping Using Padlock Probes and Rolling Circle Amplification

2016 ◽  
pp. 59-76 ◽  
Author(s):  
Tomasz Krzywkowski ◽  
Thomas Hauling ◽  
Mats Nilsson
Keyword(s):  
Single Molecule ◽  
Rolling Circle Amplification ◽  
Rolling Circle ◽  
Padlock Probes

scholarly journals In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

2004 ◽  
Vol 1 (3) ◽  
pp. 227-232 ◽  
Author(s):  
Chatarina Larsson ◽  
Jørn Koch ◽  
Anders Nygren ◽  
George Janssen ◽  
Anton K Raap ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Dna Molecules ◽  
Rolling Circle ◽  
Padlock Probes

scholarly journals Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

2021 ◽  
Author(s):  
Dongjian Cao ◽  
Sa Wu ◽  
Caili Xi ◽  
Dong Li ◽  
Kaiheng Zhu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Signal To Noise Ratio ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Strand ◽  
Detection Methods ◽  
Rolling Circle ◽  
High Level

Abstract Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs were used to develop novel AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

scholarly journals Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification

2011 ◽  
Vol 8 (1) ◽  
pp. 37 ◽  
Author(s):  
Sara Henriksson ◽  
Anne-Lie Blomström ◽  
Lisbeth Fuxler ◽  
Caroline Fossum ◽  
Mikael Berg ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Replicative Form ◽  
Rolling Circle ◽  
Padlock Probes ◽  
In Situ Assay

scholarly journals Direct RNA targeted transcriptomic profiling in tissue using Hybridization-based RNA In Situ Sequencing (HybRISS)

2020 ◽  
Author(s):  
Hower Lee ◽  
Sergio Marco Salas ◽  
Daniel Gyllborg ◽  
Mats Nilsson
Keyword(s):  
De Novo ◽  
Detection Efficiency ◽  
Rolling Circle Amplification ◽  
Fold Increase ◽  
Data Interpretation ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Multiple Transcripts ◽  
Detection Approach

Highly multiplexed spatial mapping of multiple transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore the possibilities of a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing (HybISS) chemistry. We benchmark a dRNA targeting kit that circumvents the standard reverse transcription limiting, cDNA-based in situ sequencing (ISS). We found a five-fold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell typing. With this increased efficiency, we maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap between the gold standard of fluorescent in situ hybridization (FISH) based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS, nor with FISH.

Rolling Circle Amplification of Cyclizable Dna Probes by ∅29 Dna Polymerase: A Tool for in Situ Single-Copy Gene Detection.

1997 ◽  
Vol 3 (S2) ◽  
pp. 201-202
Author(s):  
Paul M. Lizardi ◽  
Patricia Bray-Ward ◽  
David C. Ward
Keyword(s):  
Dna Polymerase ◽  
Rolling Circle Amplification ◽  
Single Copy ◽  
Rolling Circle Replication ◽  
Gene Detection ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Cytological Localization ◽  
High Degree

Oligonucleotide probes that can be cyclized by ligation (“padlock probes”) provide a very high degree of recognition specificity. Nilsson et al. have demonstrated the used of padlock probes for the cytological localization of alphoid repeats in chromosome 12. We have been extending the use of padlock probes to the detection of single copy sequences, and with this in mind have explored the amplification of DNA circles.We designed a primer complementary to the arbitrary backbone (non-probing) sequence of a 92-base closed circular probe oligonucleotide and investigated the kinetics of rolling circle replication. Using the highly processive, strand-displacing DNA polymerase of phage ∅29 (kindly provided by Dr. Margarita Salas, CSIC, Madrid, Spain) we demonstrated that several hundred tandem copies of the circular oligonucleotide are generated in a few minutes of incubation at 32°C. Because the amplified DNA remains hybridized to the circle in a rolling circle reaction, this method of amplification offers unique advantages for in situ gene detection since the amplified DNA can not diffuse away from the site of synthesis.

scholarly journals Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

2017 ◽  
Vol 8 (5) ◽  
pp. 3668-3675 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Yupeng Sun ◽  
Xiaojun Ren ◽  
Jinghong Li
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Rolling Circle Amplification ◽  
Robust Method ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Target Rna

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells.

Toehold-initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

2014 ◽  
Vol 126 (9) ◽  
pp. 2421-2425 ◽  
Author(s):  
Ruijie Deng ◽  
Longhua Tang ◽  
Qianqian Tian ◽  
Ying Wang ◽  
Lei Lin ◽  
...  
Keyword(s):  
Single Cells ◽  
Rolling Circle Amplification ◽  
Rolling Circle
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