title: Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes.
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.
authors.affiliation: Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Schaeck
authors.affiliation: Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium.
authors.forename: W
authors.initials: W
authors.lastname: De Spiegelaere
authors.affiliation: Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: De Craene
authors.affiliation: Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: W
authors.initials: W
authors.lastname: Van den Broeck
authors.affiliation: Department of Pharmaceutical Analysis, Laboratory of Drug Quality &Registration, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: B
authors.initials: B
authors.lastname: De Spiegeleer
authors.affiliation: Department of Comparative Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Burvenich
authors.affiliation: Department of Pathology, Bacteriology and Avian Diseases, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: F
authors.initials: F
authors.lastname: Haesebrouck
authors.affiliation: Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Decostere
mesh_terms: D000818:Animals; D001492:Bass / Q000235:genetics*; D020869:Gene Expression Profiling; D007413:Intestinal Mucosa / Q000378:metabolism*; D007814:Larva; D061346:Laser Capture Microdissection; D012313:RNA; D020871:RNA Stability; D059467:Transcriptome
chemical_list: D012313:RNA