Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad...

Area of Science:

• Molecular Biology
• Genomics
• Aquaculture

Background:

• Accurate gene expression quantification via real-time RT-PCR necessitates normalization with stable reference genes.
• The mudskipper fish Monopterus albus is an important aquaculture species, yet suitable reference genes for its gene expression studies are not well-established.

Purpose of the Study:

• To evaluate the expression stability of five candidate reference genes (18S ribosomal RNA, β-actin, glyceraldehyde phosphate dehydrogenase, elongation factor-1-α, and ribosomal protein L17) in Monopterus albus.
• To identify reliable reference genes for normalizing real-time RT-PCR data in gonadal tissues across different developmental stages and in other M. albus tissues.

Main Methods:

• Cloning of partial sequences for elongation factor-1-α (EF1α) and ribosomal protein L17 (RPL17) from Monopterus albus.
• Quantitative assessment of the expression levels of five candidate reference genes (18S, β-actin, GAPDH, EF1α, RPL17) across various M. albus tissues and gonadal developmental stages.
• Statistical analysis to determine the expression variation and suitability of each candidate gene as a reference.

Main Results:

• The expression levels of 18S ribosomal RNA, β-actin, and glyceraldehyde phosphate dehydrogenase exhibited significant variations, rendering them unsuitable as reference genes.
• Elongation factor-1-α (EF1α) and ribosomal protein L17 (RPL17) demonstrated stable expression patterns across the analyzed tissues and developmental stages.
• EF1α and RPL17 are identified as potentially reliable reference genes for normalizing real-time RT-PCR data in Monopterus albus.

Conclusions:

• Commonly used reference genes (18S, β-actin, GAPDH) are not appropriate for accurate gene expression studies in Monopterus albus.
• Elongation factor-1-α (EF1α) and ribosomal protein L17 (RPL17) are validated as suitable reference genes for quantitative gene expression analysis in Monopterus albus gonads and other tissues.
• The selection of appropriate reference genes is crucial for reliable molecular studies in aquaculture species like Monopterus albus.