Primers and probe

Primers, probes and cycling conditions as described by Stauble et al., 2014 were used [11 (link)]. A commercial rtPCR kit (AgPath-ID™ One Step RT-PCR Kit, Ambion, Austin, USA) was used as master mix according to manufacturer’s instructions, with final concentrations of 300 nM primers, 100 nM DnAprTM-vMGB, 250 nM DnAprTM-bMGB and 5 μL of template DNA. Primers and probes were synthesised commercially (Primers and probes, Applied Biosystems, California, USA) Reactions were carried out and analysed (7500 Fast Real-Time PCR System, Life Technologies) with a set threshold of 0.05. Two DNA extracts derived from sheep from flocks 1 to 14 were assayed by the rtPCR in two separate runs. Singular DNA extracts derived from sheep from flocks 15 to 24 were assayed by the rtPCR in one run.
Positive extraction controls from live cultures of isolate A198 and C305, and purified and isolated genomic DNA from the same culture isolates were used as rtPCR controls in each run. The rtPCR run was considered valid when results obtained in rtPCR controls were concordant. Results are reported as cycling threshold (Ct) values, the point at which the sample signal exceeds the threshold of 0.05. Samples producing a probe-specific fluorescent signal were defined as being positive. The effect of two cut-off values; Ct < 40 and Ct < 35, on the rtPCR detection rate and specificity, was assessed.

Total RNA was isolated from thick cryosections of murine salivary glands and cervical lymph nodes with an RNeasy mini kit (Qiagen). RNA was then reverse transcribed using the high capacity reverse transcription cDNA synthesis kit (Applied Biosystems) according to the manufacturer’s specifications. Reverse transcription was carried out on an Eppendorf Thermal Cycler PCR machine. Quantitative real-time (qRT)-PCR (Applied Biosystems) was performed on cDNA samples for ccl19, cxcl13, lta, ltb and aicda mRNA expression. β-actin was used as an endogenous control. The primers and probes used were from Applied Biosystems. qRT-PCR was run in duplicates on a 384-well PCR plate (Applied Biosystems) and detected using an ABI PRISM 7900HT instrument. Results were analysed with the Applied Biosystems SDS software (SDS V.2.3) as previously described [32 (link)].

Total RNA (1 μg) was isolated with PRImeZOL reagent (Canvax Biotech, Córdoba, Spain) and reverse-transcribed using an iScript cDNA synthesis kit (Bio-Rad, ref. 1708891). Real-time PCRs were carried out using SYBR green reagent (Bio-Rad, ref. 1725124). Alternatively, M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used to reverse-transcribe total RNA, and mRNA expression was assessed via qPCR with primers and probes from Applied Biosystems (Waltham, MA, USA), normalized to hypoxanthine-guanine phosphoribosyltransferase levels. mRNA levels were assessed on the ABI Prism7500 sequence detection system.
primers and probes for real-time qPCR:
Primers for SYBR green analysis:
GAPDHForward: 5′-ATGGGGAAGGTGAAGGTCG-3′ Reverse: 5′-GGGTCATTGATGGCAACAATATC-3′FLIP_LForward:5′-CCTAGGAATCTGCGTGATAATCGA-3′ Reverse: 5′-TGGGATATACCATGCATACTGAGATG-3′FLIP_SForward: 5′-GGGCCGAGGCAAGATAAGCAAGG-3′ Reverse: 5′-TCAGGACAATGGGCATAGGGTGT-3′CTGFForward: 5′-AGCTGACCTGGAAGAGAACA-3′ Reverse: 5′-CAGGCACAGGTCTTGATGAA-3′Taqman primers and probes:
CYR61Hs00155479_m1HPRT1Hs01003267_m1

Total RNA was isolated using the Qiagen total RNA purification kit (Qiagen, Valencia, CA, USA) and, once extracted, its integrity and concentration were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR (QRT-PCR) was performed with an ABI PRISM 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA) on 100 ng of total RNA reverse-transcribed using the High-Capacity complementary DNA (cDNA) Archive Kit (Applied Biosystems). Each cDNA sample was run in triplicate using primers and probes supplied by Applied Biosystems as pre-made solutions and the Faststart Universal Probe Master Mix (Roche Diagnostics, Mannheim, Germany) containing all the reagents necessary for amplification. Normalization of signals was obtained using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as an endogenous control. Statistical analysis of QRT-PCR results was conducted using the (2^−∆∆Ct) method, which calculates relative changes in gene expression of the considered target mRNA normalized to the endogenous control and related to a calibrator sample. The values obtained were represented in terms of relative quantity of mRNA level variations [80 (link),81 (link)].

Real time PCR was performed using TaqMan gene expression assay kits (Thermo Fisher) and the Quant Studio 7 flex real time PCR system (Thermo Fisher). Gene expression was analyzed by quantitative real‐time PCR using primers and probes (Life Technologies) for analysis of intraflagellar transport 88, IFT88 (Mm00493675_m1); cyclooxygenase‐2, COX‐2 (Mm00478374_m1) and GAPDH (4 351 309). GAPDH was used as a housekeeping gene endogenous control and relative fold change in gene expression was calculated using the 2^−ΔΔCT method.