Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis

Abstract

BackgroundBivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (ACT) is most frequently used as qRT-PCR reference gene without validation.ResultsIn this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop ( Patinopecten yessoensis ) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.ConclusionsThis work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster

“…The differences between their study and ours may be attributable to different developmental stages (D-/umbo veligers in that study vs. development from oocyte to D-veliger here) and also the different candidate gene sets (14 genes in that study vs. seven genes here). In another bivalve species, Yesso scallop (Patinopecten yessoensis), the expression of 12 housekeeping genes was analyzed in early embryos/larvae (Feng et al, 2013). Those authors also used samples from fertilized oocyte to D-veliger, but the sampling points differed considerably from ours (five stages in that study vs. 11 stages here).…”

Characterization of the <i>TRAF3IP1</i> gene in Yesso scallop (<i>Patinopecten yessoensis</i>) and its expression in response to bacterial challenge

“…The running program was as follows: 50 °C for 2 min, 94 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 62 °C for 1 min. Cytochrome B (CB) and DEAD-box RNA helicase (HELI) were used as internal reference genes for normalization of gene expression in the embryos/larvae and tissues (Ct values: CB, 16.1 ± 0.7; HELI, 25.2 ± 0.9), respectively, because they were found to be the most stably expressed reference genes in P. yessoensis embryos/larvae and tissues (Feng et al, 2013). β-actin was chosen as the internal reference gene for normalization of gene expression in the bacterial challenge experiments (Ct value: 18.9 ± 2.8), because it was found to be stably expressed in P. yessoensis hemocytes with bacterial infection and has also been widely used as a reference control for gene expression analysis in bivalves with bacterial challenge He et al, 2012;Wang et al, 2015;Wu et al, 2015).…”

Genome-wide identification and characterization of five MyD88 duplication genes in Yesso scallop (Patinopecten yessoensis) and expression changes in response to bacterial challenge

“…Specific primers were designed to amplify MyD88 cDNA fragments. Because they are the most stable genes in different developmental stages and adult tissues, cytochrome B (CB) and DEAD-box RNA helicaselike protein (HELI) were selected as reference genes for the embryo/ larva samples and tissue samples, respectively [41]. According to previous studies, scallop b-actin was used as an internal control for the immune-responsive samples [25,42,43].…”