Ionascu, A. | bioRxiv Science

bioRxiv2023

qDATA - an R application implementing a practical framework for analyzing quantitative Real-Time PCR data

Gene expression assays that are based on quantitative real-time PCR (qRT-PCR) method are still very popular, therefore, we developed qDATA, a modern open-source R based bioinformatics application that offers a quick and intuitive analysis of raw cycle threshold (Ct) values. The application relies on a straightforward specific data input consisting in Ct values and a few other mandatory fields specifying the experimental and control groups, as well as the target and house-keeping genes. Afterwards, it automatically performs descriptive statistics, normality and statistical testing on 2-{Delta}Ct (or {Delta}Ct) and 2-{Delta}{Delta}Ct terms calculated with Livaks method. Each computation is indicated in dedicated tabs that display their output consisting in dynamic data tables and publishing quality graphs. We also detail a qRT-PCR data analysis framework that depends on performing exhaustive {Delta}Ct calculations within discrete biological replicates (BRs). The Livak formula is used for the complete sets of available data. These prerequisites arguably lead to an improved data analysis and statistical relevance. In order to maximize the relevance of the results we recommend using at least three BRs and three technical replicates. The efficiency of our computing approach was tested with qDATA using input Ct values corresponding to immune related gene expression evaluated in experimental infection of Drosophila melanogaster and summer versus winter Apis mellifera workers, respectively. Our results reveal that our working strategy is very reliable and highlight the efficacy and performance of qDATA application.

genetics↗

bioRxiv2022

A novel P{lacW} insertion in the CG18135 gene of Drosophila melanogaster is linked to pupal lethality and gene overexpression in adult males

Drosophila melanogaster has been at the forefront of genetic studies and biochemical modelling for over a century. Yet, the functions of many genes are still unknown mainly because no phenotypic data are available. Herein, we present first evidence data regarding the particular molecular and other quantifiable phenotypes, such as viability and anatomical anomalies, induced by a novel P{lacW} insertional mutant allele of CG18135 gene. So far, the CG18135 functions have only been theorized based on electronic annotation and presumptive associations inferred upon high-throughput proteomics or RNA sequencing experiments. The descendants of individuals harboring the CG18135P{lacW}CG18135 allele were scored in order to assess mutant embryos, larvae and pupae viability versus Canton Special. Our results revealed that the homozygous CG18135P{lacW}CG18135/CG18135P{lacW}CG18135 genotype determines significant lethality both at the inception of larval stage and during pupal development. Few imago escapers that breach the puparium and even more rarely fully exit from it exhibit specific eye depigmentation, wing abnormal unfolding and strong locomotor impairment with apparent spasmodic legs movements. Their maximum lifespan is shorter than two days. When using the quantitative Real-Time PCR (qRT-PCR) method to confirm that CG18135 is indeed upregulated in males compared to females an unexpected gene upregulation was also detected in heterozygous mutants comparative to wild-type flies, probably because of regulatory perturbations induced by P{lacW} transposon. Our work provides the first phenotypic evidence for the essential role of CG18135, a scenario in accordance with the putative role of this gene in the carbohydrate binding processes.

genetics↗