Taqman probe

For qPCR, RNA was isolated 72 h after transfection with the RNeasy^® Mini Kit (QIAGEN, Venlo, The Netherlands). The cDNA Reverse Transcription Kit (BD Pharmingen, Franklin Lakes, NJ, USA) was used for reverse transcription. Isolation and transcription kits were utilized according to the manufacturer’s protocol.
qPCR was conducted either with a TaqMan^® Gene Expression Assay (Thermo Fisher Scientific, Waltham, MA, USA) for MSI-1, MSI-2 and NUMB, normalizing to 18S-rRNA-Expression, or with SYTO9 by using specific primers (for NOTCH-1, NOTCH-2, NOTCH-3), normalizing to HPRT expression. The results were expressed as fold change and analyzed using the 2^−∆∆ct method. TaqMan probes were obtained from Applied Biosystems (Foster City, CA, USA) while primers were from QIAGEN (Venlo, Netherlands) and Biolegio (Nijmegen, The Netherlands). Detailed information about probes and primers can be found in Supplementary Tables S2 and S3.

Species-specific primers (forward and reverse) and fluorescence-labelled probes for the multiplex qPCR assay were designed using Primer 3 software (http://primer3.sourceforge.net; Howard Hughes Medical Institute, Chevy Chase, MD, USA). The VIC and FAM labelled probes specifically target the mitochondrial cytochrome b genes of B. bovis and B. bigemina, respectively. All primers utilized in this study were synthesized through Integrated DNA Technologies (Skokie, IL, USA). TaqMan probes (VIC and FAM labelled) were synthesized through Applied Biosystems (ThermoFisher Scientific Pty Ltd., Victoria, Australia).
TaqMan qPCR was conducted in a Rotor Gene Q (QIAGEN Pty Ltd., Victoria, Australia). Reactions (20 µL) included 10 µL of SensiFAST 2× Probe Mix (Bioline Australia Pty Ltd., Alexandria, NSW, Australia), 500 nM of each oligonucleotide primer, 150 nM of the VIC- (B. bovis) or 6FAM- (B. bigemina) labelled probe, and 1 µL of extracted genomic DNA template. Temperature cycling conditions were: 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 54 °C for 30 s and 72 °C for 30 s, acquiring fluorescence on both the green (FAM) and yellow (VIC) channels at the end of each extension step. Ct scores were recorded for each sample. Negative controls (no template or DNA extraction kit buffer) were included in each PCR run.

Total RNA of ME tissue samples were extracted using Trizol (Life Technologies, Darmstadt, Germany) according to manufacturer's instructions. Sorted cells were extracted using PicoPure Isolation RNA kit (Thermo Fischer Scientific, Germany). Complementary DNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Expression of mRNA was quantified in triplicates by a RT-PCR with exon-spanning primers, Quantitect primer assay (Qiagen), or TaqMan probes (Supplementary Table 1) and SYBR Green PCR Master Mix or the TaqMan Gene Expression Master Mix (Applied Biosystems) were used for the PCR.

Tissue segments were snap-frozen, then homogenized in ice-cold RLT buffer (Qiagen, United Kingdom) containing β-mercaptoethanol (1% v/v; Sigma) using a Precellys24 instrument. RNA was extracted using RNeasy mini-prep kits (Qiagen) and gene expression levels measured using a 1-step RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) master mix (Promega, United Kingdom) and a 7500 Fast qPCR instrument (Applied Biosystems, United Kingdom) using TaqMan probes (Qiagen) recognizing Ptgis (probe ID: Mm00447271_m1), Ptgs1 (probe ID: Mm00477214_m1), Ptgs2 (probe ID: Mm00478374_m1), or the housekeeping genes 18S (probe ID: Mm03928990_g1) and Gapdh (probe ID: Mm99999915_g1). Data were analyzed by the comparative threshold method, with relative expression levels normalized to those to 18S and Gapdh and experimental control groups.

The multiplex assay was performed using CFX96 Touch system (Bio-rad, Hercules, CA, USA). Primers and TaqMan-probes (IDT DNA Technologies, Coralville, IA, USA), were manually designed using Sequencher software (Ann Arbor, MI, USA, version 5.4.6) (S1 Table). The specificity of the primers and probes was confirmed against GeneBank sequences with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Each 25μl PCR reaction mixture contained 2x real-time Quantifast mix with Rox (Qiagen), 0.8μM of each primer, 0.24μM of the TaqMan probe and 5μl of template DNA. Further details of the PCR assay are described in S2 Table. To assess clinical performance, 144 DNA samples previously tested by the M. cynos and M. canis using monoplex standard PCR assays [13 (link)] as part of the diagnostic service were analyzed using the newly developed multiplex assay and the results from both methods were compared.