Adult B. malayi worms were isolated from the peritoneal cavity of infected jirds (Meriones unguiculatus) (Mongolian gerbils) obtained from the NIAID filariasis Repository (University of Georgia, Athens GA). Male and female worms were carefully separated by size; broken worms were discarded. The worms were washed and immediately frozen at –80o C.
Worms were crushed under liquid nitrogen with a ceramic mortar and pestle and extracted in TRIzol reagent (Invitrogen, Carlsbad, CA) as previously described [11]. The accuracy of worm gender separation was assessed by PCR with B. malayi major sperm protein primers (BmMSP) and B. malayi embryo-associated fatty acid-binding protein primers (Bm-FAB-1) [10]. RNA quality was assessed with a model 2100 Bioanalyzer (Agilent, Palo Alto, CA). cDNA was synthesized from 5-7 ug each of male and female total RNA samples using 3DNA capture sequence primers (3DNA Array 350 Detection system, Genisphere, Hatfield, PA) and SuperScript II Reverse Transcriptase (Gibco BRL, Gaithersburg, MD) for each probe according to standard protocols. cDNA was concentrated by Microcon YM-30 filter (Millipore). cDNA was synthesized from two different male and female RNA samples (independently prepared as biologic replicates).
A two-step protocol was used for hybridization (3DNA Array 350 Detection system, Genisphere, Hatfield, PA). First, oligo arrays were hybridized to the cDNA probes in formamide based-hybridization buffer (MWG’s “Coverslips Hybridization buffer”) at 42C overnight and washed in a series of room temperature baths (2X SSC, 0.2% SDS; then 2X SSC, then 0.2X SSC) for 12 minutes each. Fluorescent Cy3- and Cy5- capture reagents were combined in MWG’s “Coverslips hybridization buffer” and added to each array. The arrays were incubated for 3 hours at 50C and washed as above. Each experiment consisted of a pair wise competitive hybridization of cDNA samples from female and male worms and a reciprocal dye-flip replicate. Since biological duplicates were tested, a total of four DNA microarrays were used for comparison of the two types of cDNA.
Slides were scanned immediately after hybridization on a ScanArray Express HT Scanner (Perkin Elmer, Boston, MA) to detect Cy3 and Cy5 fluorescence at 543 and 633nm, respectively. Laser power was kept constant for Cy3/Cy5 scans, and photomultiplier tube values were 69 and 60 volts, respectively. An additional scan was done for each slide with the PMT set for 54 and 46 volts. Gridding and analysis of images was performed with ScanArray software Express V2.0 (Perkin Elmer, Boston, MA). Each spot was defined on a pixel-by-pixel basis, using a modified Mann-Whitney statistical test.
[10] Michalski ML, Weil GJ. Gender-specific gene expression in Brugia malayi. Mol Biochem Parasitol 1999;104:247-57. [11] Li BW, Rush AC, Tan J, Weil GJ. Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR. Mol Biochem Parasitol 2004;137:329-337. Keywords = brugia malayi nematode worm parasite filaria filariasis Lot batch = Brugia v.1 batch 1
