Computational protocol: Effect of Low Doses (5-40 cGy) of Gamma-irradiation on Lifespan and Stress-related Genes Expression Profile in Drosophila melanogaster

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[…] For the analysis of the lifespan alterations, 150–170 individuals (males and females were kept separately) were used. Flies were transferred to a fresh medium two times a week. Dead flies were counted daily. For each experimental variant 3 biological replicates were pooled. Two control groups (one–for 5 and 10 cGy, another–for 20 and 40 cGy) for males as well as for females were used, due to the large exposure time difference (1 h 23 min and 2 h 47 min–for 5 and 10 cGy; 5 h 34 min and 11 h 8 min–for 20 and 40 cGy respectively). These replicates were merged, since flies were kept in the same conditions and the similar effects in the same variants were observed.Survival functions were estimated using the Kaplan–Meier procedure and plotted as survival curves []. Median lifespan and the age of 90% mortality were calculated. The statistical analysis of survival data was conducted using nonparametric methods. Comparison of survival functions was done using the modified Kolmogorov–Smirnov test []. The statistical significance of differences between the mean lifespans for the experimental and control variants was determined using the Gehan–Breslow–Wilcoxon test []. To test the statistical significance of differences in maximum lifespan (age of 90% mortality), the Wang–Allison test was used []. Results of the log rank test are presented in the .It is well known that the Gompertz function is applicable for describing Drosophila lifespan alterations [], so we approximate all survival curves with Gompertz equation: µ(x) = exp(αx) R0 []. We calculated parameters α and of the Gompertz equation, coefficients of determination that characterize the quality of the Gompertz function approximation [] and the mortality rate doubling time (MRDT) []. Maximum likelihood method was used to evaluate the significance of differences in the intensity of mortality []. It's well known that there is a Strehler-Mildvan correlation between α and R0 parameters of the Gompertz equation []: []: ln(R0) = γ-βα (α and R0 – parameters of Gompertz equation, γ and β –regression parameters).The Kaplan-Meier curves were plotted using STATISTICA, version 6.1 (StatSoft Inc, USA). Calculation of lifespan parameters and their statistical analysis were performed in the R software environment for statistical computing and graphics (http://www.r-project.org/). WinModest Version 1.0.2. [] was used to calculate the parameters of the intensity of mortality. [...] Real-time PCR was carried out on the 7500 Real-Time PCR System (Applied Biosystems, USA) by using modified short 6-carboxyfluorescein (FAM)-labeled probes from the Universal Probe Library (UPL, Roche, Switzerland). Pairs of primers were selected for every gene with the estimation of probability of primer dimers and heterodimers using OligoAnalyzer (http://eu.idtdna.com/calc/analyzer). The primer sequences are listed in the . Each reaction was run 3 times with 10 μL mix, containing PCR-buffer, dNTPs in concentration 250 nM, primers– 300 nM, UPL, ROX, DNA polymerase 1 unit and cDNA diluted 17.5 times. The threshold cycle Ct was determined (7500 Software v2.0.5, Applied Biosystems, USA). The amplification efficiency values were calculated as described earlier []. The primers and probes proved to be specific by electrophoresis using Bioanalyzer Agilent 2100 (Agilent Technologies, USA); the size of amplification products were as expected. [...] The first step of the analysis of qPCR data is the evaluation of the stability of reference genes by four methods ΔCT [], BestKeeper [], Normfinder [], Genorm []. The stability of all genes was analyzed relative to each other so the average rating of all genes was obtained by using all four methods. This rating showed the stability of all genes relative to each other in the certain experimental conditions. Only genes with high stability ratings were used as reference genes for expression normalization. The expression of four reference genes Actin, RpL32, EF1alpha, betaTub [] was analyzed. Analysis of expression stability revealed that genes Actin, betaTub are very variable in this experiment. So only genes RpL32, EF1alpha were used as reference for expression normalization.Ct values obtained for each gene in each sample were normalized to the reference gene Ct values for the calculation of the relative gene expression according to the formula: Ei−CtijEr1j−Ctr1j*…*Ernj−Ctrnjn, where Rij – relative gene expression of i gene in j sample, Ei, Er1j, Ernj−efficiency of reaction for gene and reference gene respectively, Ctij, Ctr1j, Ctrnj−threshold cycle of gene and reference gene respectively. All efficiencies were more than 90%. The expression change compared with control was log2FC (Fold Change), where FC = Riexp/Ricontrol for each biological replicates, then mean log2FC was calculated for all biological replicates. All calculations were performed using statistical computing programming language R (version 2.15.1). At least 2-fold mRNA level changes were considered as significant because of reference genes mRNA level variability. […]

Pipeline specifications

Software tools Statistica, OligoAnalyzer, BestKeeper, NormFinder
Applications Miscellaneous, qPCR
Organisms Drosophila melanogaster