Affymetrix GeneChip Plasmodium/Anopheles Genome Array

Differential gene expression in incipient species of Anopheles gambiae

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We examined patterns of gene expression in two independent colonies of both M and S molecular forms of Anopheles gambiae at each of three developmental stages of interest: late larvae, sugar-fed virgin females, and gravid females. For each colony, replicates were derived from independent RNA samples extracted from different cohorts to ensure that trends were reproducible.  In addition, each replicate was derived from larvae (adults) drawn from three pans (cages) to minimize the contribution of any individual pan to variation between samples. Data were obtained from a total of five biological replicates per mosquito colony.

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Transcription profiling by array of M and S molecular forms of Anopheles gambiae at each of three developmental stages

"We examined patterns of gene expression in two independent colonies of both M and S molecular forms of Anopheles gambiae at each of three developmental stages of interest: late larvae, sugar-fed virgin females, and gravid females. For each colony, replicates were derived from independent RNA samples extracted from different cohorts to ensure that trends were reproducible.  In addition, each replicate was derived from larvae (adults) drawn from three pans (cages) to minimize the contribution of any individual pan to variation between samples. Data were obtained from a total of five biological replicates per mosquito colony."

We examined patterns of gene expression in two independent colonies of both M and S molecular forms of Anopheles gambiae at each of three developmental stages of interest: late larvae, sugar-fed virgin females, and gravid females. For each colony, replicates were derived from independent RNA samples extracted from different cohorts to ensure that trends were reproducible.  In addition, each replicate was derived from larvae (adults) drawn from three pans (cages) to minimize the contribution of any individual pan to variation between samples. Data were obtained from a total of five biological replicates per mosquito colony.


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-TABM-1878

Cel files containing the raw intensity values were imported into Bioconductor (www.bioconductor.org), an open-source software project based on the R programming language (www.r-project.org).  Data quality was assessed using functions in the affy and affyPLM packages.  Due to poor quality hybridization, 3 arrays (or CEL files)were eliminated from further analysis : NB01P017.CEL, NB01P021.CEL, NB01P044.CEL). Background subtraction, normalization, and summarization of probe set data into one expression value were accomplished using the GCRMA function.  The subset of expressed genes was obtained in two steps.  First, probe sets corresponding to expressed genes were defined as those whose MAS5 normalized hybridization signals exceeded 100 for at least two of the five replicates of at least one of the four colony samples.  Second, because the Anopheles GeneChip was designed from an early genebuild (Build 2, 2003) such probe sets were re-mapped onto genebuild AgamP3.3.  Any probe sets interrogating the same gene were collapsed into a single observation, and those which did not interrogate an annotated gene were omitted.

P-TABM-1879

All laboratory colonies were maintained in the University of Notre Dame insectary under controlled conditions of 27 +/- 1 deg. Celsius, 85% RH with a 12 h:12 h light:dark cycle that included 1 h dawn and dusk transitions.Colonies were maintained in separate bays to avoid contamination.Eggs were placed in plastic trays (27 cm x 16 cm x 6.5 cm) containing 1L of R0 (reverse-osmosis purified) water.  Larvae were reared at a density of ~100 per pan and fed daily with a 2:1 mixture of finely ground tropical fish pellet:bakers yeast.

P-TABM-1880

Total RNA was isolated and purified from pools of 15-20 individuals using the RNeasy Mini Kit (Qiagen).  RNA quality and quantity were assessed using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) at wavelengths of 230, 260, and 280 nm.  The integrity of total RNA was further verified by running 200 ng samples on 1.5% agarose gels.  Total RNA (5 μg per sample) was treated with DNase I (Invitrogen, Carlsbad, CA) to remove any contaminating DNA.

P-TABM-1881

Late larvae were 4th instar larvae harvested 2-8 h prior to pupation.

P-TABM-1882

All laboratory colonies were maintained in the University of Notre Dame insectary under controlled conditions of 27 degrees C, 85% RH with a 12 h:12 h light:dark cycle that included 1 h dawn and dusk transitions.  Colonies were maintained in separate bays to avoid contamination. Eggs were placed in plastic trays (27 cm x 16 cm x 6.5 cm) containing 1L of R0 (reverse-osmosis purified) water.  Larvae were reared at a density of ~100 per pan and fed daily with a 2:1 mixture of finely ground tropical fish pellet:bakers yeast.  Pupae were transferred to 0.2 m3 emergence cages.  After emergence, adult mosquitoes were supplied absorbent cotton saturated with 20% sucrose solution.

P-TABM-1883

Virgin females were isolated by sorting newly emerged adults ( less than 6 h after emergence) into adjacent sex-specific cages; they were harvested 30 min following the onset of the dusk cycle transition on day 3 post-emergence.

P-TABM-1884

To collect gravid females, males and females were maintained in the same cage post-emergence to allow females to become inseminated.  On mid-afternoon of day 3 after emergence, females were offered a bloodmeal on a volunteer's arm; females that did not successfully engorge were removed.  An oviposition cup was placed into each cage 72 h post-bloodfeeding and females were allowed to oviposit overnight.  Egg cups were removed the following morning, and females were subject to a second bloodfeeding on a human host mid-afternoon.  Gravid females were harvested 82 h following the 2nd bloodmeal, at 4 h post-dusk. This timing, determined through independent experimental trials, represented the window during which the highest density of eggs was laid.

Transcription profiling by array of M and S molecular forms of Anopheles gambiae at each of three developmental stages

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