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

## TL;DR

This study identifies the most stable reference gene for gene expression analysis in Schistosoma japonicum during specific developmental stages post-infection.

## Contribution

The study provides validated reference genes for accurate gene expression analysis in Schistosoma japonicum at 18 and 23 days post-infection.

## Key findings

- GAPDH showed the most consistent expression at 18 and 23 days post-infection.
- TUBA was the least stable reference gene during the same time period.
- Reference gene stability varies significantly depending on the developmental stage.

## Abstract

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.