Platinum high fidelity supermix

RNA was extracted from the fat bodies of six female mosquitoes using the TRIzol method (Invitrogen) according to the manufacturer’s protocol. It was concentrated using the RNeasy MiniElute cleanup kit (Qiagen) for further processing. RNA was treated with DNase I (Invitrogen), after which cDNAs were synthesized from 2 μg of this total RNA using the Omniscript Reverse Transcriptase kit (Qiagen). PCR was performed using the Platinum High Fidelity Supermix (Invitrogen). qRT-PCR was performed using the iCycler iQ system (Bio-Rad) and an IQ SYBR Green Supermix (Bio-Rad). Quantitative measurements were performed in triplicate and normalized to the internal control of actin mRNA for each sample. Real-time data were collected and exported to Excel (Microsoft) for analysis. RNA extraction, cDNA synthesis and subsequent qRT-PCR analysis were done as previously described [15 (link)]. All the primers used are listed in S3 Table.

DNA was extracted from bacterial isolates using the Sigma Genelute Bacterial genomic DNA kit (Arklow, Wicklow, Ireland). The primers used in this study are listed in Additional file 1. Universal primers 27 F and 1492R [23 (link)] were used to amplify the 16S rRNA gene in a 50 μl reaction mixture consisting of 45 μl Platinum High Fidelity Supermix (Invitrogen, USA), each primer at 25 μM, 20 ng of template DNA and water to make the reaction up to 50 μl. Amplification conditions for the PCR included an initial denaturation step of 94°C for 2 min, followed by 35 cycles of 94°C for 20 s, 52°C for 30 s, and 68°C for 2 min and a final extension step of 68°C for 10 min. PCR products were checked for size and purity on a 1% (w/v) agarose gel using gel electrophoresis. PCR products were purified with the QIAquick PCR purification kit (Qiagen, USA). DNA sequencing of the amplified 16S rRNA gene region was carried out by Beckmann Coulter Genomics (Takely, UK). Sequence alignments were performed using the ClustalW application in BioEdit [66 (link)]. MEGA (version 5) [67 (link)] was used to construct trees by using the neighbour-joining algorithm and the Kimura two-parameter substitution model. Branch support was measured by 1,000 replicate bootstrap tests for each analysis.

cDNAs were synthesized from 2 μg total RNA using the SuperScript III Reverse Transcriptase kit (Invitrogen). RNA was treated with DNase I (Invitrogen) before cDNA synthesis. PCR was performed using the Platinum High Fidelity Supermix (Invitrogen).