Cfx 96 c1000 thermocycler

Samples of wound tissue were homogenized in Tri-Reagent using a Tissumizer rotor-stator homogenizer (Teledyne Tekmar, OH). RNA was isolated by either chloroform phase separation with an Ambion Ribopure kit (Life Technologies, NY), or directly from Tri-Reagent with a Direct-Zol miniprep plus kit (Zymo Research, CA), and subsequently reverse transcribed into cDNA using iScript supermix (Bio-Rad, CA). Genes of interest were then assayed by qPCR using mouse TaqMan probes for STAT4 (Mm00448890_m1) Ccl2 (Mm00441242_m1) and Ccr2 (Mm00438270_m1) (Life Technologies, NY) with JumpStart Taq polymerase (Sigma-Aldrich, MO), 3 mM MgCl₂, and 200 μ M dNTPs (Promega, WI) using a Bio-Rad CFX96 C1000 thermocycler. A standard TaqMan cycling protocol was performed, consisting of a 10 min 95°C hold, followed by 40 rounds of 15 sec at 95°C and 1 min at 60°C. 18s expression was used as a housekeeping control for data normalization.

DNA purification was carried out using QIAmp DNA Mini Kit (QIAGEN GmbH, Hilden, Mettmann, Germany), according to the manufacturer’s instructions, assessed for purity and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
A quantitative PCR assay was carried out using a CFX96-c1000 Thermocycler (Bio-Rad laboratories, Mississauga, ON, Canada) targeting the proteinase K (PK) gene, as described previously [30 (link),31 (link)]. The total volume per reaction was 20 µL, which included 20 nanograms (ng) of genomic DNA, the Fast SYBR Green Master Mix (Invitrogen, Burlington, ON, Canada), 10 µL of SYBR Green, 0.5 µL of forward and reverse specific primers with a final concentration of 10 picomolar (pmol)/ µL targeting the ILTV PK gene (F: 5′-TAC GAT GAA GCG TTC GAC TG -3′ and R: 5′-AGG CGT GAC AGT TCC AAA GT -3′) and DNAse/RNAse-free water (Thermo Scientific, Wilmington, DE, USA). Thermocycler conditions were 95 °C for 20 s for initial denaturation, then 40 cycles of denaturation to 95 °C for 3 s, annealing at 60 °C for 30 s and elongation at 95 °C for 10 s.

The qPCR assay was performed using Fast SYBR^® Green Master Mix (Quntabio^®, Beverly, MA, USA) with a final reaction volume of 20 µL using cDNA as a template. Each reaction volume consisted of 10 µL of SYBR Green master mix, 100 ng of respective cDNA sample, 0.5 µL of forward primer, 0.5 µL of reverse primer, and molecular biology grade water. All these qPCR assays were carried out using a CFX 96-c1000 Thermocycler (Bio-Rad Laboratories, Mississauga, ON, Canada) and the thermal cycling conditions were initial denaturation at 95 °C for 20 s, followed by 40 cycles of amplification at 95 °C for 3 s, and annealing at 60 °C for 30 s. The primers targeted the conserved IBV nucleocapsid (N) gene where the forward primer and reverse primer sequences were 5′GACGGAGGACCTGATGGTAA-3′ and 5′CCCTTCTTCTGCTGATCCTG-3′, respectively [22 (link)].

The quantification of IBV genome load in swab and tissue cDNA samples was performed with quantitative PCR (qPCR) assay using Fast SYBR^® Green Master Mix (Quntabio^®, Beverly, MA, USA). Each reaction volume incorporated 10 µL of SYBR Green master mix, 100 ng of interested cDNA sample, 0.5 µL 10 µM of forward primer, 0.5 µL 10 µM of reverse primer, and molecular biology-grade water to reach a final reaction volume of 20 µL. The qPCR assays were performed in a CFX 96-c1000 Thermocycler (Bio-Rad Laboratories, Mississauga, ON, Canada) with a thermal profile of 20 s of initial denaturation at 95 °C, 40 cycles of amplification with 3 s of denaturation at 95 °C, and 30 s of annealing at 60 °C. The forward and reverse primers used in this qPCR assay were 5′GACGGAGGACCTGATGGTAA-3′ and 5′CCCTTCTTCTGCTGATCCTG-3′, respectively, and they were targeted against the conserved IBV N gene [38 (link)].

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, 74106, Hilden, Germany) according to the manufacturer’s instructions. cDNA was prepared from RNA extracts using the PrimeScript RT Reagent Kit (Takara, RR037A, Kusatsu shi, Japan) according to the manufacturer’s instructions. The expression of selected genes was analyzed by quantitative real-time PCR performed on a CFX96 C1000 Thermocycler (BioRad) using Kapa SYBRgreen PCR master mix (Sigma, KK4617) as previously described [47 (link)]. The expression of the different analyzed genes was normalized to the expression of β-actin. The primer sequences are given in Table 1.

Total ribonucleic acid (RNA) was extracted from the tissues and swabs using Trizol^® reagent (Invitrogen Canada Inc., Burlington, ON, Canada) following the manufacturer’s recommendations. RNA concentration was measured with a Nanodrop 1,000 spectrophotometer (ThermoScientific, Wilmington, DE, United States). To synthesize the cDNA from swabs and tissues, we used 1,000 and 2,000 ng of RNA, respectively, using random primers (High-Capacity Reverse Transcription Kit^™, Applied Biosystems, Invitrogen Canada Inc., Burlington, ON, Canada). The IBV genome load quantification was performed by real-time quantitative polymerase chain reaction (RT-qPCR) test using the CFX 96-c1000 Thermocycler (Bio-Rad Laboratories, Mississauga, ON, Canada). The RT-qPCR test was performed based on a SYBR^® Green Master Mix (Invitrogen, Burlington, ON, Canada). Each reaction was adjusted to 20 μL net volume, including 10 μL of SYBR Green master mix, 100 ng of cDNA per sample, and forward and reverse specific primers (0.5 μL for each). The primers used in this RT-qPCR assay targeted the nucleocapsid (N) gene of IBV as described previously (37 (link)). In order to quantify the absolute number of IBV genome copies, a standard curve was used using six ten-fold serial dilutions (10⁷–10²) of in-house prepared plasmids bearing the IBV-N gene (37 (link)).

The CFX 96-c1000 Thermocycler (Bio- Rad Laboratories, Mississauga, ON, Canada) was used to quantify INF-γ mRNA expression. The INF-γ gene expression was quantified relative to the mRNA expression of the housekeeping gene, β-actin. The qPCR assays for both the β- actin and INF-γ genes were run on the same plate. Fast SYBR^® Green Master Mix (Invitrogen, Burlington, ON, Canada) was used. The primers for the INF-γ gene (F-ACACTGACAAGTCAAAGCCGCACA, R-AGTCGTTCATCGGGACCTTGGC) [39 (link)] and primers for the β-actin gene (F-CAACACAGTGCTGTCTGGTGGTA, R-ATCGTACTCCTGCTTGCTGATCC) [39 (link)] were used in each reaction. Each reaction volume consisted of 10 µL of SYBR Green master mix, 200 ng of cDNA of respective samples as a template, and 0.5 µL of forward and reverse specific primers that targeted the genes. The qPCR conditions were 95 °C for 20 s of pre-incubation and 95 °C for 3 s. For 40 amplification cycles, it was 60 °C for 30 s. Then, 95 °C and 65 °C with a 0.5 °C rise in temperature every 5 s was used for the melting curve analysis.