Project description:Fly strains were obtained from the Bloomington Stock Center. The chromosomes containing the gene deletion mod(mdg4)L3101 {y[1] w[1118]; P{w[+mC]=lacW}mod(mdg4)[L3101]/TM3, Ser[1]}, HP1 {In(1)w[m4h]; Su(var)205[5]/In(2L)Cy,In(2R)Cy, Cy[1]}, mod(mdg4)G16853 {w[1118]; P{w[+mC]=EP}mod(mdg4)[G16853]/TM6C, Sb[1]}, and Jil-1Scim {y[1]; P{y[+mDint2] w[BR.E.BR]=SUPor-P}JIL-1[Scim] ry[506]} were introgressed into the genetic background y[1]; bw[1]; e[4]; ci[1] ey[R] to generate the strains Mod(mdg4)/+, Su(var)205/+ and JIL-1/+. All females used in the introgression were collected within 7 hours after eclosion. For gene expression analyses, flies were grown in incubators at 25°C, 65% of relative humidity, and constant light. Newly emerged male adults flies harboring the mutations and the Y chromosomes Yohio and Ycongo(Mod(mdg4)/+;Yohio and Mod(mdg4)/+;Ycongo, Su(var)205/+;Yohio and Su(var)205/+;Ycongo, and JIL-1/+;Yohio and JIL-1/+;Ycongo) were collected and aged for 2 days at the same rearing condition before they were flash-frozen in liquid nitrogen. Four replicas of each sample were collected and stored at -80°C. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). The genetic interaction of the Y chromosome and the mutation was studied by the following contrasts: 1) male flies Mod(mdg4)/+;Yohio versus male flies Mod(mdg4)/+;Ycongo; 2) male flies Su(var)205/+;Yohio versus male flies Su(var)205/+;Ycongo; 3) male flies JIL-1/+;Yohio versus male flies JIL-1/+;Ycongo). As reference we used the same genetic background without the mutations [+/+;Ycongo (background 2) and +/+;Yohio (background 2)]. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma. Dye "swaps," loop design.

Project description:Fly strains were obtained from the Bloomington Stock Center. The chromosomes containing the gene deletion mod(mdg4)L3101 {y[1] w[1118]; P{w[+mC]=lacW}mod(mdg4)[L3101]/TM3, Ser[1]}, HP1 {In(1)w[m4h]; Su(var)205[5]/In(2L)Cy,In(2R)Cy, Cy[1]}, mod(mdg4)G16853 {w[1118]; P{w[+mC]=EP}mod(mdg4)[G16853]/TM6C, Sb[1]}, and Jil-1Scim {y[1]; P{y[+mDint2] w[BR.E.BR]=SUPor-P}JIL-1[Scim] ry[506]} were introgressed into the genetic background y[1]; bw[1]; e[4]; ci[1] ey[R] to generate the strains Mod(mdg4)/+, Su(var)205/+ and JIL-1/+. All females used in the introgression were collected within 7 hours after eclosion. For gene expression analyses, flies were grown in incubators at 25°C, 65% of relative humidity, and constant light. Newly emerged male adults flies harboring the mutations and the Y chromosomes Yohio and Ycongo(Mod(mdg4)/+;Yohio and Mod(mdg4)/+;Ycongo, Su(var)205/+;Yohio and Su(var)205/+;Ycongo, and JIL-1/+;Yohio and JIL-1/+;Ycongo) were collected and aged for 2 days at the same rearing condition before they were flash-frozen in liquid nitrogen. Four replicas of each sample were collected and stored at -80°C. Total RNA was extracted from whole flies using TRIzol (Life Technologies). The synthesis of cDNA and its labeling with fluorescent dyes (Cy3 and Cy5) as well as hybridization reactions were carried out using 3DNA protocols and reagents (Genisphere). The genetic interaction of the Y chromosome and the mutation was studied by the following contrasts: 1) male flies Mod(mdg4)/+;Yohio versus male flies Mod(mdg4)/+;Ycongo; 2) male flies Su(var)205/+;Yohio versus male flies Su(var)205/+;Ycongo; 3) male flies JIL-1/+;Yohio versus male flies JIL-1/+;Ycongo). As reference we used the same genetic background without the mutations [+/+;Ycongo (background 2) and +/+;Yohio (background 2)]. Slides were scanned using Axon 400B scanner (Axon Instruments) and GenePix Pro 6.0 software. Foreground fluorescence of dye intensities was normalized by the Loess method in Bioconductor / Limma.

Project description:Gene expression in endometrial cancer cells (Ishikawa) after short time exposure to progestins Sample and RNA preparation: The series is composed of sixteen hybridizations for analysis of differentially expressed genes in endometrial cancer cells (Ishikawa). In eight independent experiments, Ishikawa cells in monolayer received with 30 ug/ml progestin and were grown in RPMI-1640 medium (Sigma, Sty. Louis) with 10% dialyzed foetal calf serum (PAA-laboratories, Austria) without phenol red and antibiotics. Cells were seeded at a density of 1 x 105 cells/ml in 25 cm2 culture flasks in a humidified atmosphere (5% CO2) at 37°C for 4 hours. Cells not treated with progestin served as control groups. Total RNA extraction was performed using TRIZOL Reagent (GIBCO BRL Life Technologies, New York, NY). Labeling and hybridization: Total RNA was reverse transcribed and labeled with Cy3- and Cy5- attached dendrimer, respectively, using the Genisphere 3DNA 350HS kit (Genisphere, Montvale, NJ) as described in the manufacturer's protocol. Hybridizations of transcribed probes were carried out in a TECAN HS4800 instrument using the formamide-based hybridization buffer from Genisphere containing 5% dextrane sulfate and 5.5 ng/ul COT1 DNA (GIBCO BRL Life technologies) at 37º for 23 hours. 3DNA dendrimer hybridizations were carried out in formamide-based hybridization buffer alone. Post-hybridization washes were carried out at room temperature with 2xSSC for 1 min, 0.2% SDS /2xSSC for 1 min and finally with 0.2xSSC for 30 sec. Sixteen hybridizations including dye-swap have been performed for eight independent experiments. Scanning, image analysis and data processing: The arrays were scanned with the GenePix 4000B scanner (Axon Instruments Inc). The features were extracted from the arrays using GenePix Pro 6.0 (Axon instruments Inc., Union City, CA). All subsequent statistical analyses were performed using the statistical package R (R Development Core Team). Spots that displayed a signal-to-noise ratio of less than 2, or that were significantly saturated (more than 20 % saturation among foreground pixels) were filtered out. The median was used as the averaging measure of the foreground pixels. After quality control, genes that were present in less than 50 % of the arrays were filtered out. The arrays were normalized using dye-swap normalization. One array, whose dye swap twin failed was normalized using the lowess method. Statistical significance was assigned to the genes using the SAM methodology. The bioconductor SAMR package was used in the actual analysis. Missing expression levels were imputed using 10 nearest neighbour's imputation. The local FDR threshold was set to 25 %. Keywords: Progestin treatment, Control-treatment

Project description:Sample and RNA preparation: The series is composed of seventeen hybridizations for analysis of differentially expressed genes in placenta tissues from women after vaginal delivery (labouring) and after elective caesarean section (non-labouring). Total RNA extraction was performed by using the MagNa Pure Compact RNA Isolation Kit (Roche Applied Science). Labeling and hybridization: Total RNA was reverse transcribed and labeled with Cy3-attached dendrimer using the Genisphere 3DNA HS kit (Genisphere) as described in the manufacturer’s protocol. Hybridizations were carried out with a TECAN HS4800 instrument using the foramamide-based hybridization buffer from Genisphere containing 5% dextran sulphate and 0.5 microgram COT1 DNA/microgram RNA at 37°C for 23 hours. 3DNA dendrimer hybridizations were carried out in formamide-based hybridization buffer alone. Post-hybridization washes were carried out at room temperature with 2xSSC for 1 min, 0.2% SDS/2xSSC for 1 min and finally with0.2xSSC for 30 sec. Scanning, image analysis and data processing: The microarrays were scanned on an AXON 4000B scanner and images were quantified using GenePix pro 6.0 software. The background estimates were calculated using the morphological opening method. Spots that displayed a signal-to-noise ratio of less than 3 or that were significantly saturated (more than 20 % saturation among foreground pixels) were filtered out. The median was used as the averaging measure of the foreground pixels. After quality control, genes that were present in less than 50 % of the arrays were filtered out. Normalization was carried out using lowess normalization Sample labelling was balanced across the groups, presumably attenuating any dye-effect. For the purpose of finding differentially expressed genes empirical Bayes analysis, using the LIMMA package was applied. The data were analysed using a two-component linear model. The prior guess of the number of D.E genes was set to 0.01. Multiple testing was accounted for by estimating the false discovery rate. We applied the Benjamini-Hochberg procedure, as well as Storeys less conservative Q-value procedure was applied. In none of the cases were any genes significant at a reasonable (15-20% FDR) level. In summary, a 2.5-fold change in expression of any gene that was present on ≥ 8 arrays was considered significant, if the difference in fluorescence intensity between two groups reached a p-value of < 0.01. Keywords: Tissue samples of placenta from women after vaginal delivery (labouring) and after elective caesarean section (non-labouring)

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Reproductive activity triggers accelerated male mortality and decreases lifespan: genetic and gene expression determinants in Drosophila.

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