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Pooled larvae from 183 squirrels were tested.
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Also, because the mt genome of Co. oncophora was determined from pooled larvae rather than single worms, there is some potential that the sample sequenced contained one or more heterologous species of strongylid.
Total RNA was extracted from 3 dpf larvae (5 10 pooled larvae per treatment group), using Trizol reagent (Invitrogen) following the manufacturer's protocol.
The specific messengers were searched by RT/PCR in polyA+ prepared from i) salivary glands of individual adult females at day 5 of the bloodmeal, ii) pooled salivary glands at day 0 of the bloodmeal (unfed females), iii) pooled salivary glands of females at day 3 of the bloodmeal, iv) pooled nymphs and v) pooled larvae.
Pools of larvae from 2 to 22 DAH (n = 3) were collected, washed with DEPC water, frozen in liquid nitrogen and stored at -80°C until analysis.
Gene expression was measured on Nimblegen Drosophila transcriptome arrays (Nuwaysir et al. 2002) using total RNA from between three and six replicates of 30 larvae pooled from different vials.
Organ-specific mechanisms cannot be studied in pooled whole larvae, representing a methodological limitation of using RNA from whole fish larvae for microarray examinations.
Each sample contained hemolymph pooled from 30 larvae.
Hemolymph was pooled from ten larvae to obtain ∼1 μl for assays.
To ensure enough RNA was available for downstream transcriptomic analysis, 15 larvae were pooled together from each exposure unit.
Tick DNA was obtained from egg batches (ARG, AUS and MOZ strains), pooled larvae (New Caledonia strain) or pooled whole ticks (India and Indonesia strains).
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