Sybr premix extaq 2 tli rnase plus

Total RNA was extracted with TRIZOL reagent (Invitrogen). cDNA syntheses were performed with PrimeScript RT Reagent Kit (Perfect Real Time) (TaKaRa Bio). To determine the expression site, cDNA was prepared from synganglions (tick brain), salivary glands, ovaries, reproductive tracts (uterus and oviducts), midguts, fat bodies and the remaining tissues (mainly cuticle, muscles and a small amount of attached fat body) of final instar nymphs 9 days after engorgement and mated females 2 days after engorgement. Ticks have a single large gut corresponding to the midgut of insects and the whole gut was used for expression analyses. Tick fat body is a very fine tissue associated with the trachea [14 (link)], so fat bodies were collected with the trachea. To determine developmental profiling of OmSpo and OmShd expression, ovaries and midguts were used, respectively. Actin was used as an internal control [21 (link)]. Absolute quantification of OmSpo, OmShd and OmAct were performed with SYBR Premix Ex Taq II (T li RNase plus, TaKaRa Bio) on a Thermal Cycler Dice Real Time System (TaKaRa Bio). Final concentrations of all primer sets were 0.05 μM and PCR conditions were as recommended by the manufacturer. PCR reactions were performed for 30 s at 95°C, and 40 cycles at 95°C for 5 s and 60°C for 60 s. Three to four individual ticks were used for each day.

For total bacterial 16S rRNA gene, qPCR was performed using 515F and U806R primers [49 (link),50 (link)] with the thermal conditions, qPCR mixture components, and qPCR reaction conditions as previously described [48 (link)]. Arcobacter 16S rRNA gene was quantified via qPCR using 2 µL of template DNA, 12.5 µL of a MightyAmp for Real Time (SYBR Plus) (Takara Bio, Kusatsu, Japan), 0.1 µL each of 1 µM of Arco-F and Arco-R-rev primers [31 (link)], and 10.3 µL of ultrapure water. For the virulence genes ciaB and pldA, 2 µL of template DNA, 12.5 µL of a SYBR Premix Ex Taq II (Tli RNase Plus) (Takara Bio), 0.1 µL each of 1 µM of ciaB-F and ciaB-R primers (for the ciaB gene) [42 (link)] or pldA-F and pldA-R primers (for the pldA gene) [42 (link)], and 10.3 µL of ultrapure water were used. qPCR was performed with a Thermal Cycler Dice Real Time System Single TP850 (Takara Bio) under the following thermal conditions: for Arcobacter 16S rRNA gene, 98 °C for 2 min, followed by 35 cycles at 98 °C for 10 s, 55 °C for 30 s, and 68 °C for 40 s; for the ciaB gene, 95 °C for 30 s, followed by 35 cycles at 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 20 s; and for the pldA gene, 95 °C for 30s, followed by 35 cycles at 94 °C for 15 s, 56 °C for 45 s, and 72 °C for 20 s. A melting curve analysis was performed to confirm the generation of specific qPCR products.