Selection of Housekeeping Genes As Internal Controls for Quantitative RT-PCR Analysis of the Veined Rapa Whelk ()

Background: The veined rapa whelk is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in . For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in for use as internal controls for qRT-PCR.

Methods: In this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1 (), -actin (), cytochrome c oxidase subunit 1 (), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 subcomplex subunit 7 (), 60S ribosomal protein L5 (), 60S ribosomal protein L28 (), glyceraldehyde 3-phosphate dehydrogenase (), -tubulin (), 40S ribosomal protein S25 (), 40S ribosomal protein S8 (), ubiquitin-conjugating enzyme E2 (), histone H3 (), and peptidyl-prolyl cis-trans isomerase A (). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.

Results: Of the 13 candidate genes tested, we found that was the most stable internal control gene in almost all adult tissue samples investigated with and as secondary choices. For the normalization of a single specific tissue, we suggested that and are the best combination in gonad, as well as and for intestine, and for kidney, and for gill, and for Leiblein and mantle, , , and for liver, , and for hemocyte. From a developmental perspective, we found that was the most stable gene in all developmental stages measured, and and were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, , , and for stage 1, and for stage 2 and 5, and for stage 3, and and for stage 4.

Discussion: Our results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of in the future.