# Validation of reference genes for RT-qPCR relative expression analysis during cyst-to-early adult development of Taenia solium

## TL;DR

This study identifies reliable reference genes for measuring gene expression during the development of the tapeworm Taenia solium from larval to adult stages.

## Contribution

The study validates rpl13 and ef1α as the most stable reference genes for RT-qPCR in Taenia solium development.

## Key findings

- rpl13 and ef1α were ranked as the most stable reference genes across three developmental stages of Taenia solium.
- The study provides a validated framework for gene expression analysis during early adult development of the parasite.
- The identified reference genes can improve accuracy in measuring gene regulation during key developmental processes like strobilation.

## Abstract

The development of the zoonotic parasitic tapeworm Taenia solium from larval to adult involves significant but often clinically overlooked events crucial in cestode biology. The early-adult events can be studied in vitro, providing a valuable model to examine scolex evagination, strobilation, and worm development. With some transcriptomes being reported, single-gene relative expression analysis using reverse transcription of RNA (RT) followed by quantitative PCR (qPCR) is valuable to confirm differential expression and study gene regulation during parasite development. However, accurate comparisons with this approach require the validation of endogenous reference genes (RGs). This study identifies stable RGs for normalizing transcript expression data in Taenia. We examined 12 candidate RGs across three “early tapeworm” phases grown in culture. Transcripts were evaluated with RNA-seq and qPCR. Stability rankings were generated using geNorm and NormFinder (for RNA-seq) and RefFinder (for qPCR). Transcripts for rpl13 and ef1α were ranked as the most stable and were tested by using them to normalize the expression of h2b and wnt11a, involved in proliferation and strobilation processes.

Taenia solium is a parasitic tapeworm that causes taeniasis and cysticercosis, diseases that severely affect populations in low-resource regions. While the cystic larval stage in pigs is well characterized, much less is known about the parasite’s transformation into the adult tapeworm, which occurs exclusively in the human intestine. This process is critical for transmission, as adult tapeworms release eggs that spread infection. In this study, we used cysts in culture representing the early events of adult worm development. Our goal was to identify stable reference genes necessary for accurately measuring gene expression during this process. Building on our previously generated in vitro gene expression dataset focused on initial adult development, we validated key reference genes that will support future research into parasite biology and may contribute to strategies aimed at controlling its spread and reducing disease burden.