Rotor gene q rg 6000

Candidate genes identified by RNA-seq were further validated using RT-qPCR. RNA (0.5 μg) was treated with Turbo DNase (Thermo Fisher Scientific) and reverse transcribed using Oligo-dT primers and Superscript III (Thermo Fisher Scientific). Real-time PCR was performed in triplicates with SYBR green (Qiagen) using a Rotor-Gene Q RG-6000 (Qiagen). Data was normalized to 18S and presented as 2^−ΔC_T. Primer design was performed using the NCBI primer designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) on the Equus caballus (taxid:9796) genome. Primer pairs were selected based on the presence of at least one intron on the corresponding genomic DNA. Oligonucleotide primers used in qPCR are listed in Supplementary Table S2.

Quantitative analyses of the genes for α-SMA, vimentin, fibronectin, ColI, MMP7, TGF-β1, HIF-1α, Gcm1, and GAPDH were performed using real-time PCR (Rotor-Gene Q/RG-6000; Qiagen, Hilden, Germany). Total RNA was extracted from harvested kidney tissue using the TRIzol RNA isolation system (NucleoSpin^® RNA, TaKaRa Bio, Shiga, Japan). First-strand cDNA was synthesized from 1 μg of RNA using a reverse transcriptase kit (High-Capacity RNA-to-cDNA™ Kit, Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed using the Roter-Gene SYBR Green PCR Kit (Qiagen). The PCR reaction involved 40 cycles, and the conditions were as follows: 95 °C for 5 s and 60 °C for 10 s. The specific primer sequences used are mentioned in Supplementary Table S2. Primers for Wnt family were previously described^{45 (link)}. Expression of the various genes was calculated after normalization with GAPDH.

Biological triplicates of eggs and/or L1 were added 1,000 μl lysis buffer (Buffer ATL, QIAGEN, Hilden, Germany) and 20 μl of Proteinase K (20 mg/ml, QIAGEN), re-sealed with Parafilm® M (Bemis NA, Oshkosh, USA) and incubated at 56 °C for 2 h. After incubation, each sample was mixed by gentle shaking and DNA was extracted from 1/5 of the total lysis buffer (200 μl) and processed by QIAamp® DNA Mini Kit (QIAGEN) following the manufacturer’s instructions. Primers and probe targeted a 91 bp stretch in the O. ostertagi ITS2 sequence (GenBank® accession no. AB245021.2) from position 1036 according to Höglund et al. [15 (link)]. Duplicate amplifications were performed with a Rotor-gene Q RG-6000® (QIAGEN) in total volumes of 25 μl using 0.65 U Taq2000® polymerase (Agilent Technologies, Santa Clara, CA, USA), 0.3 μM of forward and reverse primer, 0.2 μM probe, 200 μM dNTP and 5.5 mM MgCl₂ using 2 μl DNA as template. The cycling conditions were 95 °C for 10 min and amplification for 50 cycles (95 °C for 15 s, 62 °C for 60 s). Semi-quantifications were performed by extrapolating cycle threshold (Ct) values to a standard curve with 2 × 10⁷, 2 × 10⁶, 10⁵, 10⁴ and 10³ molecules μl⁻¹ of a plasmid construct comprising the O. ostertagia ITS2 sequence according to Höglund et al. [15 (link)]. Positive and negative DNA controls and a water template control were included for each run.