#Overgrowth free download zip files software#

The incidence of the strong overgrowth phenotype (Class 3) is also slightly higher in male (32%) than in female (18%) larvae ( Figure 1B). Our results showed that 52% of the female larvae and 63% of the male larvae showed overgrown discs (Classes 2+3) ( Figure 1B). We have found that wing discs from late mbt-RNAi third instar larvae present different levels of overgrowth that can be assigned to one of three distinct phenotypic classes ( Figure 1A): (1) discs without any morphological anomaly (2) discs with mild effects consisting of small overgrowth, primarily in the hinge zone (3) discs with strong defects consisting of large or multiple overgrowths, not only in the hinge but also in other regions of the wing disc, such as the pouch or the notum. To this aim, we analysed discs that drive the UAS-l(3)mbt-RNAi under the Ubi-Gal4 in flies that are heterozygous for the l(3)mbt allele. Protein class annotation of those genes was conducted using the Panther web tool. Gene expression levels were considered as significant when the adjusted p-value was below 0.001 and the absolute log fold-change was greater than 1.5. Different contrasts were considered when computing limma linear models, taking into account pair-wise comparisons of male versus female. In summary, 12,043 transcripts were left for the downstream differential gene expression analysis. Here, only those probes that mapped to an annotation were kept because transcript cluster identifiers can refer to multiple gene symbols and, therefore, they cannot be unambiguously assigned. Gene annotation was retrieved for the corresponding probe identifiers of the GeneChip Drosophila Genome 2.0 Array (Affymetrix, catalogue # 520087), from the drosophila2.db R library. The row-wise medians from the expression data were computed, as they represent the transcript medians, and a median intensity cut-off was chosen based on the data histogram this resulted in 6624 transcripts excluded. A “soft” intensity-based filtering was applied as recommended by the limma user guide. As microarray data commonly show a large number of probes in the background intensity range, combining a low variance with a low intensity, they could end up being detected as differentially expressed although they are barely above the “detection” limit and are not very informative in general. Lowly expressed genes were filtered out from the resulting expression matrix. An extra quality control step was undertaken by applying the Relative Log Expression (RLE) transform. CEL files were processed with oligo package, which performed background correction using a deconvolution method, quantile normalization, and the Robust Multichip Average (RMA) algorithm for summarization. The microarray analysis protocol from R BioConductor workflows was adapted to analyse the samples.

#Overgrowth free download zip files software#

Affymetrix GeneChip Command Console software (AGCC) was used to acquire GeneChip images and generate. Washing and staining of microarrays were performed using a GeneChip Fluidics Station 450 (Affymetrix) and arrays were scanned with GeneChip scanner GSC3000 (Affymetrix). cDNA was hybridized to the GeneChip Drosophila Genome 2.0 Array (Affymetrix, (Santa Clara, CA, USA), catalogue # 520087) for 16 h at 45 ☌ in a GeneChip Hybridization oven 645 (Affymetrix). Subsequently, 8 µg of cDNA was fragmented and labelled using GeneChip Human Mapping 250K Nsp Assay Kit (Affymetrix catalogue # 900766), according to manufacturer’s instructions. Briefly, cDNA was generated from 25 ng of RNA using the TransPlex ® Complete Whole Transcriptome Amplification Kit (Sigma reference WTA2) and 17 cycles of amplification. Microarrays were performed by the IRB Functional Genomics Facility. RNA was purified using RNA Clean XP bead suspension (Agencourt Bioscience, Beverly, MA, USA), according to manufacturer’s instructions. Samples were then incubated at 65 ☌ for 15 min. Discs were homogenized using the pipette in 45 µL of lysis buffer (20 mM DTT, 10 mM Tris-HCl pH 7.4, 0.5% SDS, and 0.5 µg/µL proteinase K). For each replicate, 10 discs from larvae of 6 dAEL were dissected in Schneider’s Insect medium (Sigma, St. For expression analysis, experiments were conducted in triplicates.