Gene Expression Analysis of B. malayi

Total RNA was extracted from 2 female adult B. malayi worms for each sample using a TRIzol-based method and PureLink RNA Mini Kits with on-column DNase I treatment (Ambion), with 5 biological replicates per experimental group. cDNA synthesis was done using the SuperScript III First Strand cDNA Synthesis Kit (Invitrogen) following the manufacturer’s protocol. Expression levels of the B. malayi LHD gene (Bm3339), and Wolbachia wBm0209 and wBm0207 genes were estimated using the standard ‘ΔΔCt’ method using primers designed with the Primer Premier 4.0 program. The expression of the B. malayi histone H3 (Bm12920) [26 (link)] or Wolbachia wsp (wBm0432) genes were used as internal controls (reference genes) [27 (link)]. Ct values for each biological replicate were generated using three technical replicates. All data were analyzed using GraphPad Prism 6 and significance was determined using an unpaired t-test.

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Voronin D., Schnall E., Grote A., Jawahar S., Ali W., Unnasch T.R., Ghedin E, & Lustigman S. (2019). Pyruvate produced by Brugia spp. via glycolysis is essential for maintaining the mutualistic association between the parasite and its endosymbiont, Wolbachia. PLoS Pathogens, 15(9), e1008085.

Publication 2019

PureLink RNA Mini Kits on-column DNase I treatment SuperScript III First Strand cDNA Synthesis Kit

Adult female Biological Cdna Dnase i Expression gene Genes Histone h3 I rna Primer Prism Replicate Rna i Synthesis Trizol Wolbachia Worms

Corresponding Organization : New York Blood Center

Other organizations : New York University, University of South Florida, Florida College, Center for Global Health, New York College of Health Professions

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Variable analysis

independent variables

Experimental group (5 biological replicates per group)

dependent variables

Expression levels of the B. malayi LHD gene (Bm3339)
Expression levels of the Wolbachia wBm0209 gene
Expression levels of the Wolbachia wBm0207 gene

control variables

Total RNA extracted from 2 female adult B. malayi worms per sample
TRIzol-based method and PureLink RNA Mini Kits with on-column DNase I treatment used for RNA extraction
CDNA synthesis done using the SuperScript III First Strand cDNA Synthesis Kit
Expression of the B. malayi histone H3 (Bm12920) gene or Wolbachia wsp (wBm0432) gene used as internal controls (reference genes)
Ct values for each biological replicate generated using three technical replicates

positive controls

Expression of the B. malayi histone H3 (Bm12920) gene or Wolbachia wsp (wBm0432) gene used as internal controls (reference genes)

negative controls

No explicit negative controls mentioned

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