An approach to quantitate maternal transcripts localized in sea urchin egg cortex using RT-qPCR with accurate...

Area of Science:

• Developmental Biology
• Molecular Biology
• Marine Biology

Background:

• The sea urchin egg cortex is crucial for early development, containing essential proteins and cytoskeletal elements.
• Established methods for isolating egg cortices have been vital for studying cellular components since the 1970s.

Purpose of the Study:

• To assess the reliability of quantitative reverse transcription PCR (RT-qPCR) for analyzing maternal transcript levels within the sea urchin egg cortex.
• To identify the most stable reference genes for accurate gene expression analysis in both whole eggs and isolated cortices.

Main Methods:

• Selection and stability assessment of seven candidate reference genes (28S, Cycb, Ebr1, GAPDH, Hmg1, Smtnl1, Ubb) using five algorithms (BestKeeper, CV, ΔCt, geNorm, NormFinder).
• RNA isolation from both whole sea urchin eggs and isolated cortices.
• RT-qPCR analysis to quantify transcript levels, including the gene of interest, Diacylglycerol kinase delta 2 (Daglb-2).

Main Results:

• Gene ranking for reference gene stability varied between whole eggs and isolated cortices.
• Ubiquitin-conjugating enzyme E2 D2 (Ubb) demonstrated the highest stability across both sample types, making it the most suitable reference gene.
• RT-qPCR confirmed an increased level of Diacylglycerol kinase delta 2 (Daglb-2) transcripts in isolated egg cortices, validating its potential cortical localization.

Conclusions:

• Quantitative RT-qPCR is a feasible method for determining the cortical association of transcripts in sea urchin eggs.
• The use of Ubb as a reference gene enhances the accuracy of transcript level analysis in egg cortex studies.
• This approach provides a reliable tool for investigating the spatial distribution of maternal transcripts during early sea urchin development.