Rotor gene q real time pcr instrument

Total RNA was isolated from whole blood of control and affected dogs using QIAamp RNA Blood Mini Kit (QIAGEN, Valencia, CA). The optional on column DNase treatment was carried out to eliminate gDNA contamination. 100ng of total RNA was used for cDNA synthesis with the SuperScript III First-Strand Synthesis System for RT-PCR (Life technologies, Grand Island, NY). Primers for quantitative real-time PCR (qRT-PCR), for PR2X7, CAMKK2 and DENR were designed using Primer3Plus [68 (link)] and are shown in S6 Table. Semi quantitative RT-PCR using AmpliTaq Gold® (Life Technologies, Grand Island, NY) was performed to confirm product size and sequence identity was confirmed by sequencing. PCR was performed in triplicates using the Rotor-Gene SYBR Green PCR Kit (QIAGEN). A 2 step cycle protocol (35 cycles; 95°C; Annealing- 15 seconds at 60°C; Extension- 90 seconds at 60°C; Final Melt curve) was carried out on the Rotor Gene Q real-time PCR instrument (QIAGEN). Each replicate containing 0.2ng template cDNA. All data were normalized to the housekeeping gene B2M, using published primer sequences [69 (link)]. Amplification efficiency and differences in takeoff values between affected and unaffected dogs were analyzed by REST2009 [70 (link)]. Box plots of calculated delta-delta CT values were generated using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA).

Real-time PCR was performed using SYBR Premix DimerEraser kit gene according to the manufacturer’s instructions (Takara, Shiga, Japan). All the reactions were carried out on a Rotor-Gene Q real-time PCR instrument (Qiagen Inc., Germany) according to the manufacturers‘ instructions. Briefly, 5 μl of SYBR Premix, 5 pmol of each primer and 50 ng of cDNA as template were used in a final volume of 10 μl. The amplification reactions were thermally cycled as follows: denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 10 s, and extension at 72°C for 15 s. Human beta-2-microglobulin (B2M) (33 (link)) was selected as a normalization standard and fold change in expression of each target mRNA relative to B2M was calculated based on 2^−ΔΔCtrelative expression formula. The primer sets were designed in exon-junction or between two adjacent exons separated by a large intron to ensure the amplification of RNA and not genomic DNA. The primers used for detecting the expression of genes are listed in table 1.

The input DNA concentration was 1 ng/µL except where noted in specificity studies, in which a serial dilution of standard DNA was prepared from 10 to 0.01 ng/µL and the indicated quantity was inputted. The DNA was amplified using a Rotor-Gene Q real-time PCR instrument (Qiagen, Germantown, MD, USA) using the following settings: 95 °C for 2 min initial denaturation, 29 cycles of 95 °C for 10 s denaturation, 66 °C for 10 s primer annealing, and 72 °C for 10 s primer extension detecting HRM. For the C. difficile assay, the following temperature program was used: 95 °C for 2 min initial denaturation, 40 cycles of 95 °C for 20 s denaturation, 62 °C for 20 s primer annealing with touchdown of 2 cycles at 0.5°, and 72 °C for 20 s primer extension detecting HRM. Melt analysis was performed using the following settings directly after amplification: 65 to 95 °C melt increasing by 0.2 °C in 2 s intervals detecting HRM.

Real-time PCR of TLR4 and OPN was performed using SYBR Green RealMasterMix (Invitrogen) according to the manufacturer's instructions, with a Rotor-Gene Q Real-time PCR instrument (Qiagen, Hilden, Germany). The primers used in this study were shown in Table 2, using β-actin as an internal control. The amplification scheme was: incubation for 5 min at 95°C, followed by 40 cycles of 10 s at 95°C, 15 s at 59°C and 20 s at 72°C, with a final extension step of 7 min at 72°C. Data were normalized to β-actin expression in each sample. In all cases, each PCR was performed with triplicate samples. Relative expression of mRNA was determined using the 2^(-ΔΔCt) method.