Kipryushina YO et al. (2022), An approach to quantitate maternal transcripts ...

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PLoS One 2022 Jun 16;176:e0260831. doi: 10.1371/journal.pone.0260831.

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

Kipryushina YO , Maiorova MA , Yakovlev KV .

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The sea urchin egg cortex is a peripheral region of eggs comprising a 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 was developed in 1970s. Since then, 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 was aimed to estimate the 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, the transcripts of which are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) based on calculated level of stability in both eggs as well as isolated cortices. Our results showed 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, we selected Daglb-2 as a gene of interest, which transcripts are potentially localized in cortex according to transcriptome analysis, and observed increased level of Daglb-2 in egg cortices by RT-qPCR. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to determine cortical association of transcripts in sea urchin eggs.

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	Fig 1. Isolated cortices from unfertilized eggs.(A) Phase contrast image of isolated cortices attached to poly-L-lysine treated coverslip. (B) DIC image of cortex at high magnification. Granular pattern indicates multiple cortical granules. (C) Confocal image of actin staining. There is small punctate pattern of actin staining, which is typical for egg cortices. Scale bars 50 μm (A), 10 μm (B, C).
	Fig 2. Boxplot of Ct values for candidate reference genes in all samples.(A) Ct values in cDNA samples synthesized from total RNA (B) Ct values in cDNA samples synthesized from mRNA. The boxes show interquartile range (25–75%), horizontal lines represent medians. The whiskers show the minimum and maximum values. No outliers were detected.
	Fig 3. Stability analysis of candidate reference genes performed by different methods.Stability estimated by BestKeeper (A), CV (B) ΔCt (C), geNorm (D) and NormFinder (E).
	Fig 4. Determination of minimal number of reference genes for accurate normalization.Pairwise variations (Vn/n+1) were calculated by geNorm in all samples (eggs and isolated egg cortices) for total RNA (A) and mRNA (B).
	Fig 5. Comparison of the normalized relative levels of Daglb-2 between eggs and isolated cortices.(A) To normalize values, three most stable genes and best pair of genes were selected. Also, data were normalized to least stable gene (28S). (B) Relative levels of Daglb-2 normalized to Ubb. P-values indicate statistical significance between columns based on t-test.