A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

Abstract

The sea urchin egg cortex is a peripheral region of eggs consisting of cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos has been developed in 70s of 20th Century. Since that time this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study is an estimation of reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb, Ebr1, GAPDH, Hmg1, Smtnl1 and Ubb, which transcripts are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, {Delta}Ct, geNorm and NormFinder) upon calculated level stability in both eggs and isolated cortices. Our results show that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices by RT-qPCR, we selected Daglb-2 as a gene of interest, which transcripts potentially localized in cortex, and found increased level of Daglb-2 in egg cortices. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to approve cortical association of transcripts in sea urchin eggs.