Validating the classics: Accurate reference gene panel for reliable RT-qPCR in Porifera

Area of Science:

• Marine Biology
• Molecular Biology
• Genomics

Background:

• Quantitative real-time PCR (RT-qPCR) is vital for gene expression analysis.
• Accurate normalization in RT-qPCR relies on stable reference genes.
• Non-model organisms like sponges present challenges due to variability and limited resources.

Purpose of the Study:

• To identify and validate stable reference genes for RT-qPCR in the calcareous sponge Leucosolenia corallorrhiza.
• To assess the cross-species applicability of reference genes in distinct sponge species.
• To provide a validated panel of reference genes for Porifera research.

Main Methods:

• Seven candidate housekeeping genes were selected based on transcriptomic and genomic data.
• Expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder in L. corallorrhiza and three other sponge species.
• Pairwise variation analysis was used to determine the optimal number of reference genes for normalization.

Main Results:

• RPL13A, ACT1, and GAPDH were identified as the most stable reference genes in Leucosolenia corallorrhiza.
• While similar gene subsets showed stability across species, no single gene was universally stable.
• Using two to three validated reference genes is recommended for accurate normalization in sponge RT-qPCR studies.

Conclusions:

• Reference gene stability in sponges is species-specific and cannot be reliably predicted by phylogenetic or ecological factors.
• A robust panel of candidate reference genes is provided for Porifera, requiring species- and condition-specific validation.
• The study highlights the need to consider technical challenges in non-model organism research.