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

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

Hao Song et al. PeerJ.

Abstract

Background: The veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa. 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 R. venosa 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-1α (EF-1α), α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1α subcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). 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 EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α, RL5, and NDUFA7 for liver, GAPDH, PPIA, and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA, RS25, and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB 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 R. venosa in the future.

Keywords: Development; Internal control; Rapana venosa; Real-time PCR; Tissue.

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Clusters of genes in different developmental samples.
Six developmental stages in triplicates consisting of one-spiral whorl stage (C), two-spiral whorl stage (D), early three-spiral whorl stage (F), late three-spiral whorl stage (G), four-spiral whorl stage (J), and juveniles (Y).
Figure 2
Figure 2. Ranking of candidate housekeeping genes in adult tissue.
NormFinder (stability value, filled squares) and GeNorm (average expression stability (M value) of remaining genes, open rhombus) ranking of candidate housekeeping genes in eight tissues together and separately. A lower value indicates more stable expression.
Figure 3
Figure 3. Ranking of candidate housekeeping genes in developmental stages.
NormFinder (stability value, filled squares) and GeNorm (average expression stability (M value) of remaining genes, open rhombus) ranking of candidate housekeeping genes in five developmental stages together and separately. A lower value indicates more stable expression.
Figure 4
Figure 4. Determination of the number of reference genes required for accurate normalization.
Pairwise variation by GeNorm between candidate genes in tissues (black bar) and developmental stages (white bar).

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Grant support

The research was supported by the Project supported by the National Natural Science Foundation of China (Grant No. 31572636), the NSFC-Shandong Joint Fund for Marine Science Research Centers (Grant No. U1606404), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11020703), the Agricultural Major Application Technology Innovation Project of Shandong Province. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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