Due to absence of transgenic approaches in Notopthalmus Viridescens (newt), and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress unique genes from newt. Novel newt genes cloned, and inserted at attP site in Drosophila were misexpressed ubiquitously using tubulin Gal-4. Sample (total RNA) for RNA sequencing was collected at 3rd instar larval stage during which major developmental events takes place in Drosophila. Total RNA was extracted, and purified using RNA clean and ConcentratorTM. RNA quality was quantitated by calculating absorbance at 260 nm (A260) and 280 nm (A280) wavelengths using Nanodrop 2000 spectrophotometer. Good quality samples had A260/ A280 ratio greater than 2 and a peak at 260 nm. Our results show that following this protocol high quality of RNA was obtained. These high quality RNA samples were used for downstream processes e.g. Next generation RNA sequencing. Of the total 36,099 transcripts in Drosophila, 34,967 transcripts were detected, and 2775 transcripts were significantly regulated by misexpressing foreign gene (Unique gene from newt) in Drosophila . Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. Wingless/Wnt was one of the important evolutionarily conserved pathway that was differentially regulated.
Copyright © 2019, the Authors
Mehta, Abijeet Singh; Luz-Madrigal, Agustin; Li, Jian-Liang; Tsonis, Panagiotis A.; and Singh, Amit, "Total RNA Extraction from Transgenic Flies Misexpressing Foreign Genes to Perform Next Generation RNA Sequencing" (2019). Biology Faculty Publications. 251.