Strains: Susceptible strain (SUS), Refractory strain (REF) Gender: female Age: 4-5 days Tissue: midgut Time: 3-96 hours after sucrose meal
Treatment protocol
Virus serotype: Dengue-2, JAM1409 virus strain in vitro cultures were maintained in the Ae. albopictus C6/36 cell line. Mosquito treatment: Cohorts of 30 starved Aedes aegypti aged 4-5 days were transferred to 500 ml paper cups were exposed to a dengue-2 infected or naïve blood meal, using standard artificial membrane feeders. Fully engorged females were provided a 5% sucrose solution ad libitum and maintained at 26oC and relative humidity. A cohort of 30 mosquitoes received a 5% sucrose meal (SM).
Extracted molecule
total RNA
Extraction protocol
Samples of at least 15 midguts were dissected at 3, 6, 12, 24, 48, 72, and 96 hours post blood meal (infected or naive) and sucrose meal. Total RNA was isolated using TRIZOL l® Reagent (Invitrogen, Carlsbad, CA).
Description
Recombinant SAGE plasmid-pZERO clones were purified by Eppendorf 96-well plasmid purification system and single pass sequencing was done on an ABI 3730xl DNA sequencer (Applied Biosystems, Foster city, CA)
Data processing
Individual SAGE ditags were extracted from the raw ABI sequences with a software created for SAGE analysis: SAGE 2000 4.5 Software, Version E (Invitrogen). The software parses out individual tags, discards ambiguous sequences, cleaves redundant ditags and generates a table of the individual tags and their observed occurrences within the library.
