Hwang H et al. (2024), A fractionation-based protocol to investigate R...

XB-ART-60523

STAR Protoc 2024 Jan 15;51:102830. doi: 10.1016/j.xpro.2023.102830.

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A fractionation-based protocol to investigate RNA solubility phase transition during Xenopus oocyte maturation.

Hwang H , Ma M , Yang J .

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While RNAs are soluble in vitro, their solubility may be altered when incorporated into some protein complexes inside the cell. The solubility phase transition of RNAs is thus indicative of changes in the function and activity of RNAs. Here, we present a protocol for the assessment of RNA solubility phase transition during Xenopus oocyte maturation. We describe steps for sample preparation, cell fractionation, RNA extraction, real-time PCR, and analysis of the obtained results. For complete details on the use and execution of this protocol, please refer to Hwang et al. (2023).1.

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Species referenced: Xenopus laevis
Genes referenced: ccna1 cep152 ddx25 dnd1 sybu wee2
GO keywords: oocyte maturation

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References [+] :

Heasman, Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules. 1991, Pubmed, Xenbase

	Graphical abstract
	Figure 1. Cell fractionation procedure for separation into soluble and insoluble fractions (A) Collection of four oocytes in a tube. (B) Homogenization of samples with a micro pestle. (C) Image of cell fraction after centrifuge. (D) Supernatant (soluble) and pellet (insoluble) fractions. (E) Image after resuspending pellet.
	Figure 2. Assessment of the integrity of isolated RNAs The gel electrophoresis image shows 18S and 28S rRNA from soluble and insoluble RNAs.
	Figure 3. Representative results of fractionation RT-qPCR (A) The percentage distribution of S-I RNAs, including wee2.S, ccna1.S, and cep152.L, in the soluble and insoluble fractions (Table 2). (B) The percentage distribution of I-S RNAs, including ddx25, sybu, and dnd1, in the soluble and insoluble fractions (Table 3).

	Figure 1. Cell fractionation procedure for separation into soluble and insoluble fractions(A) Collection of four oocytes in a tube.(B) Homogenization of samples with a micro pestle.(C) Image of cell fraction after centrifuge.(D) Supernatant (soluble) and pellet (insoluble) fractions.(E) Image after resuspending pellet.
	Figure 2. Assessment of the integrity of isolated RNAsThe gel electrophoresis image shows 18S and 28S rRNA from soluble and insoluble RNAs.
	Figure 3. Representative results of fractionation RT-qPCR(A) The percentage distribution of S-I RNAs, including wee2.S, ccna1.S, and cep152.L, in the soluble and insoluble fractions (Table 2).(B) The percentage distribution of I-S RNAs, including ddx25, sybu, and dnd1, in the soluble and insoluble fractions (Table 3).