On column dnase 1 treatment

Relative quantification of gene expression was determined by real-time RT-PCR. The expression of SMAD3 and SMAD7 genes was quantified using TaqMan Gene Expression assays as described previously [1 (link)]. Briefly, total RNA was isolated from freshly cut sections of cryopreserved jejunum previously embedded in OCT compound using the Purelink FFPE RNA isolation Kit protocol (Life Technologies). The total RNA was purified using RNA Clean and concentrator-25 Kit (Zymo Research, Irvine, CA, USA) with on-column DNase I treatment (Life Technologies) followed by RNA quantification using the Synergy H4 reader (Biotek, Winooski, VT, USA). Superscript III first-strand synthesis kit (Life Technologies) was used to synthesize cDNA from total RNA. TaqMan gene expression assays Rh02621726_m1, Hs00706299_s1, and Hs00178696_m1 (Life Technologies) were employed for the quantification of TGF-β, SMAD3, and SMAD7 transcripts, respectively, using an ABI 7900HT Fast PCR System (ThermoFisher Scientific). The expression of each gene was normalized against 18S rRNA expression using TaqMan 18S rRNA Endogenous Control Assay (Life Technologies). Relative gene expression was determined among different groups using the comparative threshold cycle (C_T) method, and fold changes in the expression were evaluated using 2^−ΔΔCT [33 (link)].

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.