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.
Highlights
The increasing demand for a sustainable aquaculture industry has promoted research regarding optimal environmental and nutritional parameters so as to prevent diseases[1]
It is generally agreed that the use of multiple internal reference genes, which were previously validated to be stably expressed in the specific experimental conditions, offers the most optimal method for reverse transcription quantitative polymerase chain reaction (RT-qPCR) normalization[16,17]
The best results were depicted for the snap frozen tissue sections with ribonucleic acid (RNA) quality indicator (RQI) values ranging from 7.0 to 9.1
Summary
The increasing demand for a sustainable aquaculture industry has promoted research regarding optimal environmental and nutritional parameters so as to prevent diseases[1]. Studies resorting to gene expression analyses in fish larvae remain scarce, and most of these studies employ homogenized whole larval bodies or segments for RNA extraction. This is rooted in the small size of fish larvae in the first weeks following hatching, which does not allow to www.nature.com/scientificreports/. LCM followed by RT-qPCR to assess gene expression in intestinal tissue has not been optimized yet in fish larvae. Intestinal tissues are known to maintain the poorest RNA integrity with fast degradation, hampering the downstream applications such as gene expression analysis[13]. It is generally agreed that the use of multiple internal reference genes, which were previously validated to be stably expressed in the specific experimental conditions, offers the most optimal method for RT-qPCR normalization[16,17]
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