Optimal Reference Genes for Gene Expression Analysis of Overmating Stress-Induced Aging and Natural Aging in Male Macrobrachium rosenbergii

Functional gene expression is closely linked to an organism's physiology and can be quantified using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). However, the stability of reference gene expression is not absolute, which may impact the accuracy of RT-qPCR results. In this study, we evaluated the suitability of nine genes including receptor for activated protein kinase c1 (<i>rack1</i>), ribosomal protein L6 (<i>rpl6</i>), ribosomal protein L9 (<i>rpl9</i>), ribosomal protein S2 (<i>rps2</i>), ribosomal protein S18 (<i>rps18</i>), ribosomal protein lateral stalk subunit P0 (<i>rplp0</i>), eukaryotic translation elongation factor 1β (<i>eef1b</i>), eukaryotic translation initiation factor 4a (<i>eif4a</i>), eukaryotic translation initiation factor 5a (<i>eif5a</i>) analyzed from RNA sequencing (RNA-Seq) data in addition to three genes including eukaryotic elongation factor 1α (<i>eef1a</i>), β-actin (<i>actb</i>), and glyceraldehyde 3-phosphate dehydrogenase (<i>gapdh</i>) selected from the literature to obtain the best internal controls in the RT-qPCR analysis of <i>M. rosenbergii</i> under overmating stress and natural aging. RefFinder was used to comprehensively evaluate the stability of the candidate reference genes. The initial results showed that three genes (<i>eif5a</i>, <i>rps18</i>, and <i>rplp0</i>) from the RNA-Seq data had relatively stable expression levels, which were more stable than those of the three commonly used reference genes. <i>Eif5a</i> and <i>rps18</i> were the best combination for the RT-qPCR analysis of <i>M. rosenbergii</i> under overmating stress and aging. Further analysis indicated that <i>eif5a</i> might be the best reference gene for the study of <i>M. rosenbergii.</i>