Recent Advances in High-sensitivity &lt;i&gt;In Situ&lt;/i&gt; Hybridization and Costs and Benefits to Consider When Employing These Methods

Higo, Shimpei; Ishii, Hirotaka; Ozawa, Hitoshi

doi:10.1267/ahc.23-00024

Cited by 5 publications

(2 citation statements)

References 41 publications

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“…It has become widely used in recent years for a wide range of applications and is beginning to be used to aid in clinical diagnosis through automation. 7 Accessibility to this technology may become easier due to its innately simplified equipment use, steps, and interpretation.…”

Section: Discussionmentioning

confidence: 99%

“… 1 In situ hybridization (ISH) is a method being used for fungal identification and as such, refinements on the technique have been developing. 3 , 4 , 5 , 6 , 7 We present a case of dermatophyte abscess caused by T rubrum detected in formalin-fixed paraffin-embedded tissue using conventional fungal stains and compared with a novel RNA—ISH technique.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A case of dermatophyte abscess and adjunctive use of a novel RNA: In situ hybridization to confirm Trichophyton rubrum

Bernales-Mendoza,

Shimizu,

Mochizuki

et al. 2024

JAAD Case Reports

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A case of dermatophyte abscess and adjunctive use of a novel RNA: In situ hybridization to confirm Trichophyton rubrum

Bernales-Mendoza,

Shimizu,

Mochizuki

et al. 2024

JAAD Case Reports

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Problems with Paralogs: The Promise and Challenges of Gene Duplicates in Evo-Devo Research

Deem,

Brisson

2024

Integrative and Comparative Biology

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Gene duplicates, or paralogs, serve as a major source of new genetic material and comprise seeds for evolutionary innovation. While originally thought to be quickly lost or non-functionalized following duplication, now a vast number of paralogs are known to be retained in a functional state. Daughter paralogs can provide robustness through redundancy, specialize via sub-functionalization, or neo-functionalize to play new roles. Indeed, the duplication and divergence of developmental genes have played a monumental role in the evolution of animal forms (e.g. Hox genes). Still, despite their prevalence and evolutionary importance, the precise detection of gene duplicates in newly sequenced genomes remains technically challenging and often overlooked. This presents an especially pertinent problem for evolutionary developmental biology (evo-devo), where hypothesis testing requires accurate detection of changes in gene expression and function, often in non-traditional model species. Frequently, these analyses rely on molecular reagents designed within coding sequences that may be highly similar in recently duplicated paralogs, leading to cross-reactivity and spurious results. Thus, care is needed to avoid erroneously assigning diverged functions of paralogs to a single gene, and potentially misinterpreting evolutionary history. This perspective aims to overview the prevalence and importance of paralogs and to shed light on the difficulty of their detection and analysis while offering potential solutions.

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Cassini: Streamlined and Scalable Method for in situ profiling of RNA and Protein

Lapique,

Kim,

Thom

et al. 2024

Preprint

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In the expanding field of spatial genomics, numerous methods have emerged to decode biomolecules in intact tissue. Advanced techniques based on combinatorial decoding can resolve thousands of features in a reasonable time but are often constrained by either the prohibitive costs associated with commercial platforms or the complexity of developing custom instruments. Alternatively, sequential detection methods, like single-molecule FISH, are easier to implement but offer limited multiplexing capability or signal amplification. Here, we introduce Cassini, a new approach for straightforward, cost-effective multiplexed measurements of mRNA and protein features simultaneously. Cassini leverages rolling circle amplification (RCA), known for its robust amplification and remarkable stability even after intense stripping, to serially detect each feature in under 20 minutes of total experimental time. The method also enables simultaneous immunostaining with either fluorophore-conjugated or DNA-barcoded antibodies, through an optimized immunostaining buffer. In a single overnight run, we show that Cassini can quantify 32 features (comprising both RNA and proteins) with sensitivity similar to state-of-the-art FISH techniques. We provide a comprehensive protocol alongside an online probe-design platform (cassini.me), aiming to enhance accessibility and user-friendliness. With our open-source solution, we aspire to empower researchers to uncover the nuances of spatial gene expression dynamics across diverse biological landscapes.

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Recent Advances in High-sensitivity <i>In Situ</i> Hybridization and Costs and Benefits to Consider When Employing These Methods

Cited by 5 publications

References 41 publications

A case of dermatophyte abscess and adjunctive use of a novel RNA: In situ hybridization to confirm Trichophyton rubrum

A case of dermatophyte abscess and adjunctive use of a novel RNA: In situ hybridization to confirm Trichophyton rubrum

Problems with Paralogs: The Promise and Challenges of Gene Duplicates in Evo-Devo Research

Cassini: Streamlined and Scalable Method for in situ profiling of RNA and Protein

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