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http://dx.doi.org/10.12750/JARB.34.4.280

Assessment of Suitable Reference Genes for RT-qPCR Normalization with Developmental Samples in Pacific Abalone Haliotis discus hannai  

Lee, Sang Yoon (Department of Marine Bio-Materials and Aquaculture, Pukyong National University)
Park, Choul-Ji (Genetics and Breeding Research Center, National Institute of Fisheries Science)
Nam, Yoon Kwon (Department of Marine Bio-Materials and Aquaculture, Pukyong National University)
Publication Information
Journal of Animal Reproduction and Biotechnology / v.34, no.4, 2019 , pp. 280-291 More about this Journal
Abstract
Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.
Keywords
abalone Haliotis discus hannai; developmental samples; reference genes; RT-qPCR assay;
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