Lightcycler 96 real time pcr machine

In order to verify the reproducibility and repeatability of gene expression data obtained by RNA-Seq, seven genes were selected for QRT PCR verification. In brief, cDNA was generated from total RNA using the PrimeScript II 1 st Strand cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer’s instruction. Quantitative RT-PCR analysis was carried out with the cDNA using SYBR green on a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland). The b-Actin was used as a reference gene for the standardization of the results. The relative expression levels were calculated as described previously (15 (link)). Three biological repeats were measured for each sample. The primers used were shown in Table 2.

Samples were initially screened using bovine and human specific primers developed by Gunathilaka et al. [20 (link)]. These primers were selected based on the abundance of available host species in the area. The reaction conditions consisted of a 10 µl reaction including 0.5 M of forward and reverse primers (Integrated DNA Technologies, Leuven, Belgium), 5 µl of SYBR green master mix (Roche, Welwyn Garden City, UK), 2 µl of nuclease-free water (Roche) and 2 µl of template DNA. PCR was run on a LightCycler 96 real-time PCR machine (Roche) under the following cycling conditions: pre-incubation of 95 °C for 600 s, 40 cycles of 95 °C for 10 s, 62 °C for 10 s and 72 °C for 30 s followed by a melting analysis.
Human-positive blood meals (including any potentially mixed feeds) from the above assay were confirmed using the Promega Plexor^® HY Human DNA forensic detection kit (Promega, Southampton, UK). Assay was performed following manufacturer’s protocol using a Stratagene MX3005P (Agilent Technologies, Santa Clara, USA) real-time PCR machine.

For qRT-PCR, ZFL cells were seeded in six-well plates at 1.2 × 10⁶/well. Each treatment was replicated three times. After treatment, the cells were washed twice with PBS, and 1 mL TRIzol reagent (Life Technologies Inc., Thermo Fisher Scientific, Waltham, MA, USA) was added to extract total RNA, according to the manufacturer’s instructions. RNA integrity was assessed using agarose gel electrophoresis and an Implen NanoPhotometer (Implen Inc., Westlake Village, CA, USA). After removing DNA from the total RNA, cDNA was synthesized using the PrimeScript^® RT Reagent Kit (TaKaRa, Otsu, Japan). qRT-PCR assays were performed in triplicate using the LightCycler^® 96 real-time PCR machine (Roche, Basel, Switzerland) in a 20 μL reaction system containing 2.0 μL primers (2.5 μM), 2.0 μL diluted first-strand cDNA, 10 μL 2× Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific), and 6.0 μL sterilized double-distilled water. The cycling conditions were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, and finally, 60 °C for 1 min. After PCR, melting curves were analyzed over a range of 72–95 °C (at 1 °C/20 s steps) to confirm product singularity. Relative gene expression levels were determined using the comparative CT method (2^−ΔΔCt), as described previously [35 (link),36 (link)].

Total RNA was extracted from AVICs using the TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA), quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and reverse transcribed into cDNA with iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Gene-specific primers used for the qPCR analysis were: human-KL Forward: 5′-CTCGGGTACCTGGTGGC-3′; human-KL Reverse: 5′-ACACCTGACCTCCCTGAGTG-3′. Quantitative qPCR was performed using a LightCycler ^® 96 Real-Time PCR machine (Roche Diagnostic, Basel, Switzerland) using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The expression of mRNA was calculated as the relative ratio to human GAPDH mRNA, with all data presented as fold change against control.

LAMP-OSD reaction mixtures were prepared in 25 μl volume containing indicated amounts of human glyceraldehyde-3-phosphate dehydrogenase (gapd) DNA templates along with a final concentration of 1.6 μM each of BIP and FIP primers, 0.4 μM each of B3 and F3 primers, and 0.8 μM of the loop primer. Amplification was performed in 1X isothermal buffer (NEB) (20 mM Tris-HCl, 10 mM (NH4)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, pH 8.8) containing 0.8 M betaine, 0.8 mM dNTPs, 2 mM additional MgSO₄, 16 units of pure Bst 2.0 DNA polymerase, and 100 nM of OSD reporter. Reporters were prepared for use in LAMP assays by annealing 100 nM fluorophore-labeled OSD strands with a 5-fold excess of the quencher-labeled OSD strands by incubation at 95°C for 1 min followed by cooling at the rate of 0.1°C/sec to 25°C). In some LAMP-OSD assays commercial Bst 2.0 was substituted either with 16 units of commercial Bst-LF DNA polymerase or with 3 μl (2 x 10⁷ cells) of Bst-LF expressing lyophilized BL21 cellular reagents (rehydrated prior to use in 30 μl water). For real-time signal measurement these LAMP-OSD reactions were transferred into a 96-well PCR plate, which was incubated in a LightCycler 96 real-time PCR machine (Roche, NC, U.S.A.) maintained at 65°C for 90 min. Fluorescence signals were recorded every 3 min and analyzed using the LightCycler 96 software.

Chicken and cattle macrophages were infected with opsonized bacteria as described above. At 6 h post-infection, infected and uninfected cells were collected, and total RNA was isolated by using the RNeasy Mini kit (Qiagen, Denmark) according to the manufacturer's instructions. RNA degradation and purity were assessed by agarose gel electrophoresis and Nanodrop analysis, respectively. After being treated with DNase following the manufacturer's instructions (Promega, Denmark), RNA was transcribed into cDNA using the GoScript™ Reverse transcription system (Promega, Denmark) following the manufacturer's instructions. Transcripts were then assessed by qPCR analysis, which was performed using the SYBR FastStart Essential DNA Green master kit (Roche, Denmark) on the Roche LightCycler 96 Real-Time PCR machine. The housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. The relative gene expression of cytokines [interleukin (IL)1β, IL6, IL10, LITAF, interferon (IFN)-γ, IL18, CXCLi1, and CXCli2] and Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) (primer sequences listed in Supplementary Tables 1, 2) was calculated and analyzed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Quantitative real-time PCR was performed on a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland) using SuperReal PreMix Plus SYBR-Green (Tiangen Biotech, China). qRT-PCR was conducted as described previously (Shen et al., 2016 (link)), and the relative expression levels were calculated as described previously (Livak and Schmittgen, 2002 (link)). The experiments were performed using three biological replicates. All the primers used in the qRT-PCR are listed in Supplementary Table 1.