Dataset Information

Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

ABSTRACT: Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.

SUBMITTER: Langenbacher AD

PROVIDER: S-EPMC4308483 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

Langenbacher Adam D AD Rodriguez Delany D Di Maio Alessandro A De Tomaso Anthony W AW

Genesis (New York, N.Y. : 2000) 20140917 1

Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in ...[more]

PMID: 25179474

Similar Datasets

Project description:BackgroundDetection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.ResultsIn this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.ConclusionBy employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.

Dataset Information

Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

Publications

Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets