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.

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 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

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.

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

scholarly journals Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

2018 ◽  
Vol 64 (12) ◽  
pp. 1704-1712 ◽  
Author(s):  
Felix Neumann ◽  
Iván Hernández-Neuta ◽  
Malin Grabbe ◽  
Narayanan Madaboosi ◽  
Jan Albert ◽  
...  
Keyword(s):  
Seasonal Influenza ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Clinical Samples ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Diagnostic Assays ◽  
Padlock Probes ◽  
Probe Assay ◽  
Digital Quantification

Abstract BACKGROUND Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants—A(H1N1), A(H3N2), B/Yamagata, and B/Victoria—and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct. RESULTS The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

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