Computational protocol: The transcriptome of the mosquito Aedes fluviatilis (Diptera: Culicidae), and transcriptional changes associated with its native Wolbachia infection

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Protocol publication

[…] To assess the accuracy of the transcriptomic data set, six contigs were selected at random for expression analysis with RT-qPCR. Two of these contigs were indicated to have higher expression for Flu mosquitoes (AF10645; comp10645_c1_seq2 and AF10453; comp10453_c0_seq5), two had higher expression in Tet mosquitoes (AF15178; comp15178_c0_seq1 and AF14155; comp14155_c0_seq1), while two had equivalent expression between Flu and Tet mosquitoes (AF2025; comp2025_c0_seq1 and AF2041; comp2041_c0_seq1). The first four of these contigs were predicted to be differentially expressed through both the bayseq and DESeq2 analyses. Primers for these contigs were designed from the sequences generated during sequencing, which meant that they were suitable for cDNA. Primer sequences were designed using Primer 3 V0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) to have a Tm of 55–60 °C and a product size range of 80–120 bp (Additional file ).Mosquitoes from both the Flu and Tet lines were reared as described above, and then females were collected individually at 6 days post eclosion. Total RNA was extracted and quantified as described above. First strand cDNA synthesis was conducted with 1 μg of total RNA from each sample using the M-MLV reverse transcriptase assay according to manufacturer’s instructions (Promega cat: C118A). cDNAs were then diluted 1:10 in nuclease-free water and stored at -30 °C. SYBR-based PCR was used to confirm the expression of each of the test genes, with 15 samples tested per mosquito line. Each gene was quantified in duplicate for all samples using the following mix: SYBR -5 μL, forward and reverse primers (10 μM) - 0.5 μL each, sterile RNase free water - 2 μL, sample 2 μL). RT-qPCR for samples was run on a LightCycler® 96 System (Roche) using the following profile: 10 min pre-incubation at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, melt curve - 95 °C for 15 s, ramp from 60 °C to 95 °C at 1.6 °C/s. Expression values for each gene were normalised against actin1 expression, which had previously been demonstrated to be a good control gene for Ae. fluviatilis []. Mean normalised expression values for each gene were calculated using Q-Gene [], and were compared statistically between Flu and Tet mosquitoes using Mann Whiney U tests (Prism v 6.0 g, Graphpad). […]

Pipeline specifications

Software tools baySeq, DESeq2, Primer3
Application qPCR
Organisms Plasmodium gallinaceum, Aedes aegypti
Diseases Infection, Malaria, Sprains and Strains, HIV Infections