Identification and Validation of Tissue-Specific Housekeeping Markers for the Amazon River Prawn Macrobrachium...

Background/Objectives: The selection and validation of species-specific housekeeping genes (HKGs) have become increasingly common in functional genomics, with application of quantitative Polymerase Chain Reaction (qPCR) or cDNA-based qPCR (RT-qPCR). Despite the Macrobrachium amazonicum having RNA-seq studies available, there are still no data on the most stable and consistent HKGs for use in relative gene expression analyses. Therefore, the present study aimed to identify and validate seven HKGs in M. amazonicum: Eukaryotic Translation Initiation Factor (EIF), 18S ribosomal RNA (18S), Ribosomal Protein L18 (RPL18), β-actin, α-tubulin (α-tub), Elongation Factor 1-α (EF-1α), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). Methods: The HKGs were identified in the M. amazonicum transcriptome, characterized for identity confirmation, and compared against public databases. Subsequently, RT-qPCR assays were prepared using muscle, hepatopancreas, gills, testis, androgenic gland, and ovary to assess the stability of the HKG markers, employing the comparative ∆Ct, BestKeeper, NormFinder, and GeNorm methods. Results: All candidate HKGs identified showed high similarity with other decapods. Reactions performed with these markers demonstrated high specificity, PCR efficiency, and elevated coefficients of determination. The comprehensive ranking, indicated that no single HKG was stable across all tissues, with HKGs showing the best stability being tissue-specific. The most stable HKGs were RPL18 and 18S. GAPDH, historically used as an HKG, showed the poorest performance in stability ranking for most tissues tested, whereas β-actin was most suitable only for ovarian. Conclusions: These data reinforce the need for species-specific HKG validation and provide an appropriate panel of reference markers for gene expression studies in the M. amazonicum.