Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR

Abstract

Improved understanding of the biology of reproduction in filarial worms may lead to identification of new targets for drugs or vaccines. Real-time RT-PCR is increasingly being adopted for RNA quantification and genetic analysis. Candidate gender-regulated genes were selected from genes identified in prior studies by differential display RT-PCR and by electronic selection of the Brugia malayi expression sequence tag (EST) database for clusters with possible gender-specific expression (four or more transcripts in male cDNA library ESTs but none in female ESTs or vice versa). Expression of candidate genes in male and female worms was compared by real-time reverse transcription PCR with sequence-specific primers. Double stranded DNA product was measured by SYBR Green I fluorescence; melting curves and agarose gel electrophoresis were used to verify the specificity of results. Relative gene expression results were normalized by parallel studies with internal control genes that were shown to be equally expressed in male and female worms (beta actin 2B, histone H3, NADH dehydroxygenase subunit 1) and calculated by the comparative C t method. Nineteen of 31 candidate genes were verified to have reproducible, gender-biased expression with fold differences between 5 and >30,000. These included several well-known genes (for example, genes encoding major sperm protein and a microfilaria sheath protein) and many novel genes. This paper reports the first large scale use of real time RT-PCR to quantitate and study gene expression in a nematode parasite. Our results represent an important step toward improved understanding of the molecular biology of reproduction in filarial nematodes.

Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cDNA oligonucleotide array analysis

“…Worms (usually 30 female and 100 male adult worms per batch) were crushed under liquid nitrogen with a ceramic mortar and pestle and extracted in TRIzol reagent (Invitrogen, Carlsbad, CA) as previously described [12]. The accuracy of worm gender separation was assessed by RT-PCR with B. malayi major sperm protein primers (BmMSP) and B. malayi embryo-associated fatty acid-binding protein primers (Bm-FAB-1) [11].…”

“…Selected gender-regulated gene candidates from the microarray analysis were chosen for confirmation studies by real-time RT-PCR as previously described in detail [12]. Briefly, complementary DNA primers (primer sequence information is available upon request) were designed from EST sequences obtained from GenBank with Primer Express software (Version 1.0, PE Applied Biosystems, Foster City, CA).…”

“…Experimental design and relative quantification calculations were carried out according to RQ software (Relative Quantification (RQ) ABI Prism 7000 Sequence Detection System). Nicotinamide adenine dinucleotide dehydrogenase subunit 1 (BMC02280, NADH subunit 1) was used as a not gender-regulated internal control [12].…”

“…The large expressed sequence tag (EST) database and extensive genomic sequence information available for B. malayi [7][8][9] provide a solid foundation for studying the molecular biology of this organism using gene expression profiling and functional genomics approaches [10][11][12][13]. We have previously identified a small number of sex-regulated genes in B. malayi worms by differential display RT-PCR and real-time RT-PCR [11,12].…”

“…We have previously identified a small number of sex-regulated genes in B. malayi worms by differential display RT-PCR and real-time RT-PCR [11,12]. One of these genes (microfilaria sheath protein) was recently shown to be essential for normal production of microfilaria by RNA interference (RNAi) [13].…”