Computational protocol: The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae)

Similar protocols

Protocol publication

[…] Quantitative real-time RT-PCR (qRT-PCR) was performed in a CFX96 Real-Time Detection System (Bio-Rad) using the Quantimix Easy Kit (Biotools, Spain). Genes encoding actin and the 26S ribosomal subunit were used as endogenous reference controls to analyze the relative transcriptional activity of selected target genes: EcR, ERR, E74, Vtg, hsp70, hsp40, cyp18a1, GAPDH, cyp4g, GPx, GSTd3, its2, rpL4, and rpL13. Primer sequences and efficiencies are shown in . The qRT-PCR was run using the following conditions: 30 s initial denaturation at 95°C, followed by 35 cycles of 5 s denaturation at 95°C, 15 s annealing at 58°C and 10 s elongation at 65°C. To verify the accuracy of each amplicon, a melting curve analysis was performed after amplification. CFX Manager 3.1 software (Bio-Rad) was used to determine total mRNA levels by normalizing the expression (2−ΔCq) of the target genes against the two endogenous reference genes. Each sample was run in duplicate wells, and three independent replicates were performed for each experimental condition. [...] Statistical analyses were performed using SPSS Statistics 22 software (IBM, USA). Normality and homoscedasticity of data were assessed with Shapiro-Wilk’s test and Levene’s test, respectively. Normalized levels of transcripts were analyzed with ANOVA, followed by Games Howell’s or Bonferroni’s post hoc tests, when appropriate. The Kruskal-Wallis’ test was used when data were not homogeneous or normally distributed, and the differences between pairs were established using Mann-Whitney’s tests. A probability (P) value ≤0.05 was used as a cutoff for statistical significance (*) of differences between treatments and control samples. […]

Pipeline specifications

Software tools Biotools, CFX Manager, SPSS
Applications Miscellaneous, Population genetic analysis
Organisms Drosophila melanogaster