Taqman universal master mix kit

Levels of mRNA were analyzed by real-time, quantitative PCR. RNA was isolated by Trizol (Life Technologies). Reverse transcription was performed with SuperScriptIII reverse transcriptase (Life Technologies). Gene-specific primers and probes were designed and bought from Taqman (Life Technologies). All real-time PCR reactions were performed using the ABI 7500-Fast Real-Time PCR System and TaqMan Universal Master Mix Kit (Life Technologies). The relative quantitative analysis of gene expression was calculated as the 2-▵▵Ct method. For each data point, at least duplicated wells were used. GAPDH was used for housekeeping gene control. And each experiment was repeated at least 3 times.

Total RNA was isolated by TRIzol (Life Technologies). Reverse transcription was performed with SuperScript III reverse transcriptase (Life Technologies). Gene-specific primers and probes were designed and bought from Taqman (Life Technologies). All real-time PCR reactions were performed using the ABI 7500-Fast Real-Time PCR System and the Taqman Universal Master Mix Kit (Life Technologies). The relative quantification of gene expression was calculated using the ΔΔCt method. We use predesigned real-time PCR primers from Applied Biosystems for the analysis of Opg (Tnfrsf11b; Mm0043545_m1), Rankl (Tnfsf11; Mm00441908_m1), AEP (Lgmn; Mm01325350_m1), GAPDH (Gapdh; Mm99999915_g1).

Total RNA was extracted using a TRIzol reagent (Invitrogen, California, USA) according to the manufacturer's instructions. Reverse transcription was performed with SuperScript III reverse transcriptase (Life Technologies, #18080085), and primers were designed and purchased from TaqMan: CEBPB (Hs00270923_s1), Cebpb (Mm00843434_s1), SNCA (Hs00240906_m1), Snca (Mm01188700_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), TNF-α (Mm00443258_m1), and TGFβ (Mm01178820_m1). Real-time PCR was performed with a TaqMan Universal Master Mix Kit (Life Technologies, #4304473) by ABI 7500 Fast Real-Time PCR System. The relative quantification of the target genes was calculated by the comparative cycle threshold (CT) (2^−ΔΔCT) method. The expression of GAPDH was used as an endogenous control. The relative quantification of gene expression was calculated by the 2-^ΔΔCT method. All tests were performed in triplicate [29 (link)].

miRNAs and total RNAs were extracted using Trizol and were cleaned using miRNeasy kit (Qiagen, Valencia, CA, USA). miRNAs were measured using Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix Kit (Applied Biosystems, Carlsbad, CA, USA). All SYBR-based real-time PCRs were run on a CFX96 or CFX384 Real-Time PCR machine (Bio-Rad, Hercules, CA, USA) with iScript reverse transcription kit and iTaq supermix (Bio-Rad). A list of SYBR-based real-time PCR primers can be found in the Supplementary Table.

For CDV vaccine strain diagnosis, the primer pair used was (CDV Vaccine Fw 5′-ATAATGATGTTATCATCAGYGATGAT-3′ and CDV Vaccine Rv 5′-CTTGGTCCGATAATGATCAACC-3′) together with the TaqMan Probe (CDV probe AM1 5′-FAM-CTTAGTAGCAYTGCCCAAGATCCCTTGATC-BHQ1-3′), designed in a 249 bp portion of the CDV M gene and M-F intergenic region of the Onderstepoort vaccine strain. This Real Time PCR protocol was developed to specifically identify the following vaccine strains: Onderstepoort, Duramune, Snyder Hill, Nobivac [15 (link)]. For the Real Time PCR vaccine CDV strains, the TaqMan^® Universal Master Mix kit (Applied Biosystems™, ThermoFisher Scientific) was used with the following composition: 12.5 µL of TaqMan^® 2X Universal PCR Master Mix, 600 nM of forward primer, 600 nM of reverse primer, 300 nM of probe, 5 µL of cDNA and 5.75 µL RNase-free water for a 25 µL total final volume. The Real Time PCR for vaccine CDV strains was carried out using Quant Studio 7 Flex (Applied Biosystems) and the conditions were: 50 °C for 2 min, 95 °C for 10 min, and 50 cycles of 95 °C for 15 s, 58 °C for 30 s and 60 °C for 1 min. All data were analysed using the Quant Studio 7 Flex Detection System SDS software package (Applied Biosystems, Foster City, CA, USA). All the examined samples were also tested in parallel with the Wilkes protocol to exclude any positivity from being linked to vaccinations.