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PloS one2014; 9(6); e100810; doi: 10.1371/journal.pone.0100810

Post-mortem stability of RNA in skeletal muscle and adipose tissue and the tissue-specific expression of myostatin, perilipin and associated factors in the horse.

Abstract: Obesity, a major concern for equine welfare, is highly prevalent in the leisure horse population. Skeletal-muscle and adipose tissues are important determinants of maintenance energy requirements. The myostatin and perilipin pathways play key roles in the regulation of muscle mass and lipolysis respectively and have both been associated with obesity predisposition in other mammalian species. High quality samples, suitable for molecular biology, are an essential prerequisite for detailed investigations of gene and protein expression. Hence, this study has evaluated a) the post-mortem stability of RNA extracted from skeletal-muscle and adipose-tissues collected under commercial conditions and b) the tissue-specific presence of myostatin, the moystatin receptor (activin receptor IIB, ActRIIB), follistatin and perilipin, genes and proteins across a range of equine tissues. Objectives were addressed using tissues from 7 Thoroughbred horses presented for slaughter at a commercial abattoir; a) samples were collected at 7 time-points from Masseter muscle and perirenal adipose from 5 minutes to 6 hours post-mortem. Extracted RN was appraised by Optical Density analysis and agarose-gel electrophoresis. b) Quantitative real time PCR and Western Blotting were used to evaluate gene and protein expression in anatomically-defined samples collected from 17 tissues (6 organs, 4 skeletal muscles and 7 discrete adipose depots). The results indicate that, under the present collection conditions, intact, good quality RNA could be extracted from skeletal-muscle for up to 2 hours post-mortem. However, RNA from adipose tissue may be more susceptible to degradation/contamination and samples should be collected no later than 30 minutes post-mortem. The data also show that myostatin and ActRIIB genes and proteins were almost exclusively expressed in skeletal muscle. The follistatin gene showed a more diverse gene expression profile, with expression evident in several organs, adipose tissue depots and skeletal muscles. Perilipin gene and protein were almost exclusively expressed by adipose tissue.
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Publication Date: 2014-06-23 PubMed ID: 24956155PubMed Central: PMC4067385DOI: 10.1371/journal.pone.0100810Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research explores how obesity in horses affects the expression of specific genes and proteins in skeletal muscle and adipose tissue. The gene pathways of myostatin and perilipin, associated with muscle mass regulation and fat breakdown respectively, were focused on. Using horses presented for slaughter, their tissues were collected and analyzed at different time points after death to study RNA stability and expressional presence of targeted genes and proteins.

Sample Collection and Analysis

  • The team addressed their research objectives using tissues collected from seven Thoroughbred horses presented for slaughter at a commercial abattoir.
  • Samples for the first part of the study were taken at seven different time points from the Masseter muscle and perirenal adipose, ranging from five minutes to six hours post-mortem.
  • The extracted RNA from these samples was then evaluated by Optical Density analysis and agarose-gel electrophoresis to study the post-mortem stability of the RNA.

Evaluation of Gene and Protein Expression in Different Tissues

  • The second part of the study involved a more detailed investigation into gene and protein expression across a variety of equine tissues.
  • Samples were collected from 17 tissues, including six organs, four skeletal muscles, and seven discrete adipose depots.
  • Using quantitative real time PCR and Western Blotting, the researchers were able to assess the gene and protein expression levels in these anatomically-defined samples.

Findings and Implications

  • Findings from the first part of the study showed that under the conditions of the study, good quality RNA could be extracted from skeletal muscle for up to two hours post-mortem.
  • However, the team found that RNA from adipose tissue may be more susceptible to degradation or contamination, and therefore samples should ideally be collected within 30 minutes post-mortem.
  • As for gene and protein expression, the team found that myostatin and ActRIIB genes and proteins were almost exclusively expressed in skeletal muscle.
  • Follistatin gene showed a more diverse expression profile, being evident in several organs, adipose tissue depots and skeletal muscles.
  • Finally, perilipin gene and protein were almost exclusively expressed by adipose tissue.

Cite This Article

APA
Morrison PK, Bing C, Harris PA, Maltin CA, Grove-White D, Argo CM. (2014). Post-mortem stability of RNA in skeletal muscle and adipose tissue and the tissue-specific expression of myostatin, perilipin and associated factors in the horse. PLoS One, 9(6), e100810. https://doi.org/10.1371/journal.pone.0100810

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 9
Issue: 6
Pages: e100810
PII: e100810

Researcher Affiliations

Morrison, Philippa K
  • University of Liverpool, Department of Obesity and Endocrinology, Faculty of Health and Life Sciences, Leahurst Campus, Neston, Wirral, United Kingdom.
Bing, Chen
  • University of Liverpool, Department of Obesity and Endocrinology, Faculty of Health and Life Sciences, Leahurst Campus, Neston, Wirral, United Kingdom.
Harris, Patricia A
  • Equine Studies Group, WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, Melton Mowbray, Leicestershire, United Kingdom.
Maltin, Charlotte A
  • University of Liverpool, Department of Obesity and Endocrinology, Faculty of Health and Life Sciences, Leahurst Campus, Neston, Wirral, United Kingdom.
Grove-White, Dai
  • University of Liverpool, Department of Obesity and Endocrinology, Faculty of Health and Life Sciences, Leahurst Campus, Neston, Wirral, United Kingdom.
Argo, Caroline McG
  • University of Liverpool, Department of Obesity and Endocrinology, Faculty of Health and Life Sciences, Leahurst Campus, Neston, Wirral, United Kingdom.

MeSH Terms

  • Activin Receptors, Type II / genetics
  • Activin Receptors, Type II / metabolism
  • Adipose Tissue / metabolism
  • Animals
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Female
  • Follistatin / genetics
  • Follistatin / metabolism
  • Gene Expression Regulation
  • Genes, Essential
  • Horses / genetics
  • Male
  • Muscle, Skeletal / metabolism
  • Myostatin / genetics
  • Myostatin / metabolism
  • Organ Specificity / genetics
  • Perilipin-1
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Postmortem Changes
  • RNA Stability
  • Real-Time Polymerase Chain Reaction
  • Software
  • Spectrophotometry

Conflict of Interest Statement

Patricia A. Harris is employed by one of the funders of this research (WALTHAM Centre for Pet Nutrition, Melton Mowbray, Leicestershire, United Kingdom). The authors can confirm that they have adhered to all the PLOS ONE policies on sharing data and materials. The authors also confirm that none of the authors record a conflict of interest.

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