*4.3. Analysis of Gene Expression*

Developing ovaries collected at 10 DAF were used for gene expression analysis. RNA was extracted using the cetyl trimethyl ammonium bromide method from approximately 6–7 frozen ovaries. First strand cDNA was synthesized from 5 ng of total RNA using the Primer Script RT Reagent kit (PR037A; Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. Quantitative

RT-PCR was performed with the LightCycler system (Light Cycler 480, Roche Diagnostics, Basel, Switzerland) using Universal Probe Library (UPL, Roche Diagnostics, Basel, Switzerland). *GAPDH* was used as the reference gene, and the relative expression levels were calculated using the 2−ΔΔCT method [37]. The primers and probes are listed in Table 4.


**Table 4.** List of primer and probes used in the gene expression analysis.
