Dig labelled sense and antisense cdna probes

In situ hybridization was performed to confirm the expression site of HaEXPB2 as previously described with some modifications69 (link). The specific primers of HaEXPB2 were used to synthesise digoxigenin (DIG)-labelled sense and antisense cDNA probes (Roche Diagnostics, Mannheim, Germany) by asymmetric PCR. Hybridization signals within the nematodes were detected by alkaline phosphatase immunostaining and specimens were observed with an Olympus IX71 microscope.
Total RNA was isolated from pre-parasitic J2, parasitic J2, J3, J4, adult females and eggs using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. All total RNA samples were treated with RNase-free DNase1 to remove DNA contamination. First-strand cDNA synthesis was conducted using the SuperScript™ III First-Strand Synthesis System for RT-PCR kit (Invitrogen). The primers GAPDH-qS1/GAPDH-qAS1 targeted the endogenous control gene GAPDH-170 (link). The PCR reactions were run using SYBR Premix ExTaq (TaKaRa, Japan) in an ABI Prism 7500 instrument (Applied Biosystems, USA). Quantification of the relative changes in gene expression was performed using the 2^−ΔΔCT method with actin as the endogenous control gene. At least three independent experiments, each with three technical replicates of each reaction, were performed.