Lightcycler probe design software

For detection of the E2 and e2 allele variants, a SimpleProbe assay was created.
The PCR primers E2for (5′-TGCAACCCCACTACAGCCT-3′) and E2Rev (5′-GAGGCAGAGCCAAAGCCTAT-3′) were designed from the Williams 82 sequence as described by Watanabe et al. 2011 [12 (link)]. This 222-bp amplicon includes the A/T SNP (Gm10:44,732,850, W82 Glyma v1.0) at base 1561 in exon 10 where a nonsense mutation occurs in the e2 allele [12 (link)]. The SimpleProbe oligonucleotide (Fluorescein-SPC-GGCATGTCTTATGAAAATATTTGCTGC-Phosphate) was designed to the E2 sequence on the sense strand using the LightCycler Probe Design software (Roche Applied Science, Indianapolis, IN). PCR reactions and melting curve parameters were identical to the E1 genotyping assay except that the forward E2for and reverse E2Rev primers had a concentration of 0.2 μM and 0.5 μM, respectively. The melting curve from 55 °C to 77 °C distinguished the E2 peak at 64 °C and the e2 peak at 60 °C.

For detection of the long juvenile trait C-del in the PI 159925 version of ELF3 (Glyma.04G050200, Wm82.a2.v1), a SimpleProbe assay was created. The primers Cdelfor (5′-TGTTCTGCAGAGAATGCGGT-3′) and Cdelr (5′- CCTCCTCCACAACCAGTTCC-3′) produce a 254-bp PCR product that contains the C/− SNP described at position 4,077,102 (Lu et al. 2017). The SimpleProbe oligonucleotide (5′-Fluorescein-SPC-GACGGTAGCCACCTTTCAAAATGCA-Phosphate-3′) was designed on the sense strand using the LightCycler Probe Design software (Roche Applied Science, Indianapolis, IN). PCR was identical as the Dt1/dt1 R166W assay with the exception that the melting curve was from 50 to 75 °C. The C-del mutant allele peak was observed at 61 °C, and the ELF3 wildtype peak was observed at 68 °C. Heterozygous samples produced both peaks.

Primers, obtained from metabion international AG, Germany, were used in this study. For the PCR assay primers, GenBank entries were searched for the selected virulence genes sequences including ureA, ureB, cagA, oipA, hpaA, babA, dupA, and napA. The primers were designed by using Primer 3 software (Table 1). For amplification of vacA s1/s2, vacA m1/m2 genes previously published PCR primers (Table 1) were used [15 (link)–18 (link)]. For the quantitative RT-PCR assay primers, GenBank entries were searched for sequences of the genes encoding human IFN-γ, T-bet, IL-17, RORγt, FOXP3. The published sequences were aligned and primers were designed by using the LightCycler probe design software (Roche Diagnostics, Mannheim, Germany) (Table 2). A BLAST search was performed to confirm the specificity of the DNAsequences of all the primers (http://www.ncbi.nlm.nih.gov/BLAST/).

Total RNA was extracted using Trizol reagent (TaKaRa Biotechnology), and cDNA was reverse-transcribed from total RNA using a SYBR^® Premix Ex Taq^TM kit (TaKaRa Biotechnology). Each well (20 μL reaction volume) contained 10 μL Power SYBR Green PCR master mix (TaKaRa Biotechnology), 0.8 μL of each primer (final concentration 0.2 μM), 2 μL template, and 6.4 μL DNAse/RNAse-free water (Sigma-Aldrich). Three wells were used for each template as biological replicates. PCR was performed using the LightCycler^®480 System (Applied Biosystems Inc.; Thermo Fisher Scientific, Inc.). Specific primers were designed using LightCycler^® Probe Design software (Roche Diagnostics, Basel, Switzerland) and synthesized by Takara Biotechnology Co., Ltd. The sequences of primers were as follows: fibulin-4: 5′-GCTGCTACTGTTGCTCTTGGG-3′, 5′-GGGATGGTCAGACACTCGTTG-3′; E-cadherin: 5′-GGATTGCAAATTCCTGCCATTC-3′, 5′-AACGTTGTCCCGGGTGTCA-3′; N-cadherin: 5′-GTAGCTAATCTAACTGTGACCGATAAGG-3′, 5′-TTGGTTTGACCACGGTGACTAA-3′; vimentin: 5′-GCAGGAGGCAGAAGAATGGTA-3′, 5′-GGGACTCATTGGTTCCTTTAAGG-3′; Snail: 5′-TCGGAAGCCTAACTACAGCGA-3′, 5′-AGATGAGCATTGGCAGCGAG-3′; Slug: 5′-TGTGACAAGGAATATGTGAGCC-3′, 5′-TGAGCCCTCAGATTTGACCTG-3′; Twist: 5′-AGCAAGATTCAGACCCTCAAGCT-3′, 5′-CCTGGTAGAGGAAGTCGATGTACCT-3′; β-actin: 5′-CCACGAAACTACCTTCAACTCCA-3′, 5′-GTGATCTCCTTCTGCATCCTGTC-3′.