Brugia malayi transcriptomics at different life cycle stages
Brugia malayi transcriptomics at different life cycle stages
Brugia malayi adults and microfilariae were obtained from the peritoneal cavities of patently infected Mongolian gerbils (Meriones uguiculatus) by peritoneal flush with prewarmed RPMI media (Fisher Scientific, Piscataway, NJ).  Microfilariae were purified by centrifugation through Ficoll-Paque? lymphocyte isolation media (Amersham Pharmacia Biotech, Piscataway, NJ), and washed in PBS three times prior to flash freezing at -80 C.  Adult worms were separated by gender, washed three times in RPMI, and flash frozen.  Egg and embryo preparations were made by repeated cutting of 10 female worms with a scalpel to release eggs and embryos into a small volume of cold RPMI.  The sample was examined microscopically and pieces of uterine tissue were removed using watchmakers' forceps.   The sample was washed three times in cold RPMI prior to flash freezing.  Immature mf (<3 days old) were generated and purified as previously described (Griffiths KG, Mayhew GG, Zink RL, Erickson SM, Fuchs JF, Mcdermott CM, Christensen BM, Michalski ML: Use of microarray hybridization to identify Brugia genes involved in mosquito infectivity. Parasitol Res 2009, In Press).  Fourth stage larvae were isolated from gerbils 12-13 days post peritoneal infection and were processed as described for adult worms.  Third stage larvae were obtained from the NIAID-NIH Filariasis Research Reagent Resource (FR3) Center at University of Georgia, Athens, GA. Total RNA was isolated from the majority of samples using a previously described protocol (Griffiths KG, Mayhew GG, Zink RL, Erickson SM, Fuchs JF, Mcdermott CM, Christensen BM, Michalski ML: Use of microarray hybridization to identify Brugia genes involved in mosquito infectivity. Parasitol Res 2009, In Press) that combines organic extraction with Trizol LS (Invitrogen, Carlsbad, CA) and column purification (RNAqeous-Micro?, Applied Biosystems, Foster City, CA).  Samples were treated with DNase I (Ambion, Austin, TX, USA) according to the manufacturer's instructions, and the absence of background DNA was confirmed by using a portion of each sample in a PCR designed to amplify the B. malayi GPX gene. Isolation of RNA from third stage larvae often produces low yield, therefore we used a modified protocol employing homogenization of tissue combined with organic extraction in RNAzol followed by cleaning, concentration, and DNase treatment using a Zymo Research RNA column (Zymo Research Corp, Orange, CA).  For all samples RNA integrity was confirmed visually by agarose gel electrophoresis; purity and concentration were determined spectrophotometrically (NanoDrop ND-1000, ThermoFisher Scientific). Samples were stored at -80?C. Total RNA was lyophilized under vacuum for transport on dry ice to the Wellcome Trust Sanger Institute Genome Facility. Polyadenylated mRNA was purified from total RNA using oligo-dT dynabead selection followed by metal ion hydrolysis fragmentation with the Ambion RNA fragmentation kit. First strand synthesis, primed using random oligonucleotides, was followed by 2nd strand synthesis to produce double-stranded cDNA. Template DNA fragments were end-repaired with T4 and Klenow DNA polymerases and blunt-ended with T4 polynucleotide kinase. A single 3' adenosine was added to the repaired ends using Klenow exo- and dATP, to reduce template concatemerization, adapter dimmer formation and to increase the efficiency of adapter ligation. Adapters (containing primer sites for sequencing) were then ligated and fragments size-selected (200-275bp) by agarose gel electrophoresis. DNA was extracted using a Qiagen gel extraction kit protocol but with dissolution of gel slices at room temperature (rather than 50oC) to avoid heat induced bias. Libraries were then amplified by PCR, to enrich for properly ligated template strands, to generate enough DNA and to add primers for flowcell surface annealing. AMPure SPRI beads were used to purify amplified templates before quantification using a Agilent Bioanalyser chip and Kapa Illumina SYBR Fast qPCR kit.
Experimental Factor: DEVELOPMENTALSTAGE
eggs and embryos