Advanced search
1 file | 641.96 KB

Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes

Author
Organization
Abstract
The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3′/5′ integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.
Keywords
laser capture microdissection, RNA integrity, RT-qPCR, reference genes, fish larvae, sea bass, Dicentrarchus labrax, gut, gene expression, REAL-TIME PCR, HEPARG CELL-LINE, DICENTRARCHUS-LABRAX, NORMALIZATION STRATEGIES, RT-PCR, EXPRESSION, SAMPLES, QPCR, SELECTION, TOOL

Downloads

  • srep21092.pdf
    • full text
    • |
    • open access
    • |
    • PDF
    • |
    • 641.96 KB

Citation

Please use this url to cite or link to this publication:

Chicago
Schaeck, Marlien, Ward De Spiegelaere, Jurgen De Craene, Wim Van Den Broeck, Bart De Spiegeleer, Christian Burvenich, Freddy Haesebrouck, and Annemie Decostere. 2016. “Laser Capture Microdissection of Intestinal Tissue from Sea Bass Larvae Using an Optimized RNA Integrity Assay and Validated Reference Genes.” Scientific Reports 6.
APA
Schaeck, M., De Spiegelaere, W., De Craene, J., Van Den Broeck, W., De Spiegeleer, B., Burvenich, C., Haesebrouck, F., et al. (2016). Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes. SCIENTIFIC REPORTS, 6.
Vancouver
1.
Schaeck M, De Spiegelaere W, De Craene J, Van Den Broeck W, De Spiegeleer B, Burvenich C, et al. Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes. SCIENTIFIC REPORTS. 2016;6.
MLA
Schaeck, Marlien, Ward De Spiegelaere, Jurgen De Craene, et al. “Laser Capture Microdissection of Intestinal Tissue from Sea Bass Larvae Using an Optimized RNA Integrity Assay and Validated Reference Genes.” SCIENTIFIC REPORTS 6 (2016): n. pag. Print.
@article{7094933,
  abstract     = {The increasing demand for a sustainable larviculture has promoted research regarding environmental parameters, diseases and nutrition, intersecting at the mucosal surface of the gastrointestinal tract of fish larvae. The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling. This study aimed at optimizing an LCM protocol for intestinal tissue of sea bass larvae. Furthermore, a 3{\textquotesingle}/5{\textquotesingle} integrity assay was developed for LCM samples of fish tissue, comprising low RNA concentrations. Furthermore, reliable reference genes for performing qPCR in larval sea bass gene expression studies were identified, as data normalization is critical in gene expression experiments using RT-qPCR. We demonstrate that a careful optimization of the LCM procedure allows recovery of high quality mRNA from defined cell populations in complex intestinal tissues. According to the geNorm and Normfinder algorithms, ef1a, rpl13a, rps18 and faua were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass across a range of experimental settings. The methodology developed here, offers a rapid and valuable approach to characterize cells/tissues in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.},
  articleno    = {21092},
  author       = {Schaeck, Marlien and De Spiegelaere, Ward and De Craene, Jurgen and Van Den Broeck, Wim and De Spiegeleer, Bart and Burvenich, Christian and Haesebrouck, Freddy and Decostere, Annemie},
  issn         = {2045-2322},
  journal      = {SCIENTIFIC REPORTS},
  language     = {eng},
  pages        = {11},
  title        = {Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes},
  url          = {http://dx.doi.org/10.1038/srep21092},
  volume       = {6},
  year         = {2016},
}

Altmetric
View in Altmetric
Web of Science
Times cited: