RNA Isolation from Early Drosophila Larval Ovaries

Abstract

In the fruit fly Drosophila melanogaster, ovarian germline stem cells (GSCs) and their niches form during larval development. This process is poorly studied partly due to technical difficulties in isolating early larval ovaries. In addition, purifying RNA from larval ovaries proves to be more challenging than purifying it from other organs. Here we describe a technique for dissecting ovaries from early larvae and advise on how to extract RNA with maximum yield and purity. RNA isolation allows assaying gene expression in a direct and quantitative manner, which is invaluable for understanding molecular events underlying ovarian niche formation and GSC establishment.

The spatiotemporal pattern of glypican coordinates primordial germ cell differentiation with ovary development

“…Larva dissections were carried out following previous method. 19 For high RNA yield, 30-40 L3 gonads were used for one biological repeat and were repeated 3 time. RNA extraction was done using TRIzol (Invitrogen) and followed by cDNA synthesis with QuantiTect Reverse Transcription Kit (QIAGEN).…”

A transitory signaling center controls timing of primordial germ cell differentiation

“…These might be stationary cells with protrusions generated in random directions or migratory cells in the process of reorienting their motile machinery. qPCR analysis Larva dissections were carried out as described previously (Gancz and Gilboa, 2017). Extracted, whole ovaries (germline and somatic tissues) were frozen in liquid nitrogen and stored at -80 C for one day.…”

A transitory signaling center controls timing of primordial germ cell differentiation

“…Larva dissections were carried out as described previously (Gancz and Gilboa, 2017). Gonads were frozen in liquid nitrogen and stored at -80°C for one day.…”