Background: Identifying optimal housekeeping genes is essential to accurately quantify gene expression dynamics across the 18th day (male and female begin to pair) and the 23rd day (female begin to sex mature) post infection of Schistosoma japonicum, because this process involves selecting suitable housekeeping genes to ensure the reliability and accuracy of all subsequent expression analyses, thereby improving the precision of biological interpretations. Schistosoma japonicum transcriptomics reveals marked stage-dependent variation in candidate reference genes, which directly challenges the long-standing hypothesis that commonly recommended reference genes remain stably expressed throughout the 18th day and the 23rd day post-infection developmental phases and therefore emphasizes the critical need for careful selection and rigorous validation in any specific experimental context. Methods: In this study, seven widely reported genes (GAPDH, TUBA, ACTB, SOD1, TP, ND and PS) of Schistosoma japonicum were systematically validated by combining Solexa high-throughput sequence analysis with targeted qPCR experiments to identify the most suitable reference genes on the 18th day and the 23rd day post infection of Schistosoma japonicum, and the expression stability of these seven candidate genes was then comprehensively evaluated using four complementary algorithms—the ΔCT method and the GeNorm V3.5, BestKeeper, and NormFinder software applications. Results: GAPDH displayed the most consistent expression profiles, whereas TUBA exhibited the least stability, particularly at the specific time points of 18 and 23 days post infection in both paired and unpaired female Schistosoma japonicum. Conclusions: The suitability of any housekeeping gene is strongly dependent on the study’s specific context and experimental conditions. Therefore, the conclusions drawn here are explicitly limited to the developmental window of 18 and 23 days post infection. Rigorous, stage-specific validation is indispensable before reliable quantitative gene expression analyses can be performed. 

Evaluation of Suitable Reference Gene During the Development of Paired or Unpaired Female Schistosoma japonicum on the 18th and the 23rd Days Post Infection

Simple Summary The development of primordial germ cells (PGCs) and sex determination (SD) is governed by a complex interplay of genes that can be targeted to hinder sexual development in fish. Obtaining high-quality ribonucleic acid (RNA) from Atlantic salmon embryos, especially after fertilization, can be extremely challenging due to the presence of a substantial amount of yolk. The objective of this research was to extract high-quality RNA from developing salmon embryos for use in downstream applications. The study also aimed to validate different housekeeping genes (HKGs) for a quantitative polymerase chain reaction (qPCR)-based assessment of PGC and SD genes during developmental stages in salmon. We isolated RNA from the developing embryos, and we present the validation of HKGs as well as the mRNA expression levels of important PGC and SD genes during the fertilization to hatching stage. The findings of this study could prove beneficial for researchers seeking to extract high-quality RNA from salmonids. Moreover, the transcript results may offer insights not only into the function of transcripts at specific developmental stages but also in identifying the stage with the highest expression levels, during which treatment to disrupt the function of the transcripts and ultimately the growth of PGCs could be administered. Abstract The challenge in extracting high-quality RNA impedes the investigation of the transcriptome of developing salmonid embryos. Furthermore, the mRNA expression pattern of important PGC and SD genes during the initial embryonic development of Salmo salar is yet to be studied. So, in the present study, we aimed to isolate high-quality RNA from eggs and developing embryos to check vasa, dnd1, nanos3a, sdf1, gsdf, amh, cyp19a, dmrt1 and foxl2 expression by qPCR. Additionally, four HKGs (GAPDH, UB2L3, eEf1a and β-actin) were validated to select the best internal control for qPCR. High-quality RNA was extracted, which was confirmed by spectrophotometer, agarose gel electrophoresis and Agilent TapeStation analysis. UB2L3 was chosen as a reference gene because it exhibited lower intra- and inter-sample variation. vasa transcripts were expressed in all the developmental stages, while dnd1 was expressed only up to 40 d°C. Nanos3a was expressed in later stages and remained at its peak for a shorter period, while sdf1 showed an irregular pattern of mRNA expression. The mRNA expression levels of SD genes were observed to be upregulated during the later stages of development, prior to hatching. This study presents a straightforward methodology for isolating high-quality RNA from salmon eggs, and the resulting transcript profiles of significant PGC and SD genes in S. salar could aid in improving our comprehension of reproductive development in this commercially important species.

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Insights into Early Ontogenesis of Salmo salar: RNA Extraction, Housekeeping Gene Validation and Transcriptional Expression of Important Primordial Germ Cell and Sex-Determination Genes

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Insights into Early Ontogenesis of Salmo salar: RNA Extraction, Housekeeping Gene Validation and Transcriptional Expression of Important Primordial Germ Cell and Sex-Determination Genes