In situ hybridization techniques for paraffin-embedded adult coral samples

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers' ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress. The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

Original languageEnglish (US)
Article numbere57853
JournalJournal of Visualized Experiments
Volume2018
Issue number138
DOIs
StatePublished - Aug 31 2018

Fingerprint

RNA Probes
Anthozoa
Paraffin
Paraffins
In Situ Hybridization
Health
Oceans and Seas
Medicine
RNA
Visualization
Cytology
Molecular biology
Acidification
Genes
Tissue
Developmental Biology
Glass
Fluid Therapy
Invertebrates
Cell Biology

Keywords

  • Anti-sense probe
  • Coral
  • Coral reefs
  • Heat stress
  • In situ hybridization
  • Issue 138
  • RNA
  • This month in JoVE

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

In situ hybridization techniques for paraffin-embedded adult coral samples. / Traylor-Knowles, Nikki.

In: Journal of Visualized Experiments, Vol. 2018, No. 138, e57853, 31.08.2018.

Research output: Contribution to journalArticle

@article{4661393228204f00878f6bf255a52680,
title = "In situ hybridization techniques for paraffin-embedded adult coral samples",
abstract = "Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers' ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress. The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.",
keywords = "Anti-sense probe, Coral, Coral reefs, Heat stress, In situ hybridization, Issue 138, RNA, This month in JoVE",
author = "Nikki Traylor-Knowles",
year = "2018",
month = "8",
day = "31",
doi = "10.3791/57853",
language = "English (US)",
volume = "2018",
journal = "Journal of visualized experiments : JoVE",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "138",

}

TY - JOUR

T1 - In situ hybridization techniques for paraffin-embedded adult coral samples

AU - Traylor-Knowles, Nikki

PY - 2018/8/31

Y1 - 2018/8/31

N2 - Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers' ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress. The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

AB - Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers' ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress. The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

KW - Anti-sense probe

KW - Coral

KW - Coral reefs

KW - Heat stress

KW - In situ hybridization

KW - Issue 138

KW - RNA

KW - This month in JoVE

UR - http://www.scopus.com/inward/record.url?scp=85054498512&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054498512&partnerID=8YFLogxK

U2 - 10.3791/57853

DO - 10.3791/57853

M3 - Article

C2 - 30222153

AN - SCOPUS:85054498512

VL - 2018

JO - Journal of visualized experiments : JoVE

JF - Journal of visualized experiments : JoVE

SN - 1940-087X

IS - 138

M1 - e57853

ER -