RNA-Seq and qPCR Analysis of Drosophila Imaginal Discs

As starting material, 120 WID and 250 eye-antenna imaginal discs (EID) were used for RNASeq. For pdm2 gene expression analysis, WIDs from 400 nub-GAL4/+; UAS-GFP/+ flies were disaggregated. RNA from sorted cells was extracted with ZR-RNA MicroPrep Kit from Zymo Research. For L3-specific genes expression, 5 third instar larvae were frozen and RNA was extracted with Quick-RNA MiniPrep Kit, from Zymo Research. Retrotranscriptions and qPCRs were performed as described previously^{17 (link)}. For quantification of RNA amounts, standard curves of each pair of primers were performed and the efficiency of amplification was calculated. The Cts obtained from the qPCR were corrected according to the amplification efficiency of the primers. Primers used for Real-Time PCR are listed in Supplementary Table 11.

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Pérez-Lluch S., Blanco E., Tilgner H., Curado J., Ruiz-Romero M., Corominas M, & Guigó R. (2015). Absence of canonical active chromatin marks in developmentally regulated genes. Nature genetics, 47(10), 1158-1167.

Publication 2015

ZR-RNA MicroPrep Kit Quick-RNA MiniPrep Kit

Cells Eye discs Flies Frozen Gene expression analysis Genes expression Larvae Primers Real time pcr

Corresponding Organization :

Other organizations : Centre for Genomic Regulation, Universitat de Barcelona, Pompeu Fabra University, Stanford University, Universidade do Porto

Top 5 similar protocols

Variable analysis

independent variables

Nub-GAL4/+; UAS-GFP/+

dependent variables

Pdm2 gene expression
L3-specific genes expression

control variables

RNA extraction with ZR-RNA MicroPrep Kit
RNA extraction with Quick-RNA MiniPrep Kit
Retrotranscriptions and qPCRs as described previously

controls

Standard curves of each pair of primers were performed and the efficiency of amplification was calculated
The Cts obtained from the qPCR were corrected according to the amplification efficiency of the primers

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