Computational protocol: Comparative physiological plasticity to desiccation in distinct populations of the malarial mosquito Anopheles coluzzii

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Protocol publication

[…] All real-time quantitative reverse transcription PCRs (qRT-PCRs) were conducted as described in []. A total of ten genes (i.e. Actin, Rps13, Rps7, Rpl5, h3a, Cytp450, Tubulin, hsp83, EGFR, and 18s) were tested as putative housekeeping genes. Following a BestKeeper analysis [], Rps13 was selected as the reference gene, since its expression was stable in all samples, whatever the experimental condition, or the anopheline population tested. Further, mRNA expression of the two AKH genes AKH-I and AKH-II was monitored. AKH-II was identified as AKH/corazonin-related peptide (see []), and will hereafter be referred to as ACP. Specific primers (forward and reverse) for both housekeeping genes and target genes were designed using the Eprimer3 software (http://emboss.bioinformatics.nl/cgi-bin/emboss/eprimer3; Additional file : Table S1) referring to the NCBI genome “Anopheles gambiae str. PEST”.Each qRT-PCR reaction was triplicated and consisted of 6 μl of absolute Blue SYBR Green Fluor (Roche Molecular Systems Inc., California, USA), 2 μl of cDNA (25 ng.μl-1), 0.5 μl of each reverse and forward primers (10 μM), and 3 μl of RNA-free water. The cycle threshold values (Ct-values) for both reference and target genes were determined using the Light-Cycler® 480 software (Roche, Meylan, France). The average Ct value of each triplicate was used to normalise candidate gene expression levels to the geometric mean of the reference gene level using the Q-Gene software []. […]

Pipeline specifications

Software tools BestKeeper, EMBOSS
Application qPCR
Diseases Malaria
Chemicals Triglycerides