Applied biosystems 7500 fast real time pcr

Total RNA from HL-1 cells was isolated using a High Pure RNA Isolation Kit (11828665001, Roche Diagnostics GmbH, Penzberg, Germany). RNA was quantified by assessing the optical density at 260 and 280 nm (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, United States). Extracted RNA was transcribed using a High-Capacity cDNA Reverse Transcription Kit (4368814, Roche Diagnostics). Gene expression was noted using TaqMan probes (Thermo Scientific) of CD36 (Mm01135198_m1), FATP4 (Mm01327405_m1) CPT1b (Mm00487200_m1), TATA box binding protein (TBP, Mm00446971_m1), Glucuronidase beta (GUSB, Mm01197698_m1) and was assessed by Applied Biosystems™ 7500 Fast Real-Time PCR (Applied Biosystems, Boston, United States). TBP and GUSB were used as endogenous controls. Data are presented as the relative gene expression change comparing target genes to the geometric mean expression of endogenous controls using the 2^–ΔCT method.

In the present study, we have included DR4:A>C (rs20576), G>A (rs6557634); FAS-1377G>A (rs2234767); FASL-844T>C (rs763110); DCC:C>G (rs2229080), A>G (rs4078288), C>T (rs7504990), A>G (rs714); PSCA:C>T (rs2294008), G>A (rs2978974); ADRA2A-1291C>G (rs1801253); ADRB1 1165C>G (rs1800544); ADRB3 190T>C (rs4994); and CYP17 T>C (rs2486758) SNPs.
Salting out method was used to isolate genomic DNA from 5 mL peripheral blood leukocytes [60 (link)]. The genotyping was performed by the PCR restriction fragment length polymorphism and TaqMan^® allelic discrimination assays (Applied Biosystems 7500 Fast Real-Time PCR (Thermo Fisher Scientific, Walthan, MA, USA)) method, as described previously [8 (link),9 (link),10 (link),11 (link),12 (link)]. PCR mix without DNA sample was taken as negative control and the 10% of random samples were sequenced to confirm the results consistency.

The amplification reactions were carried out in an Applied Biosystems 7500 Fast Real Time PCR (Thermo Fisher Scientific) using TaqMan Universal or SYBR Green mix. TaqMan and SybrGreen primers are specified in Supplemental Table 1. The comparative C_T-method was used to determine the amount of target, normalized to endogenous reference 18S or TBP and relative to a calibrator (2^−ΔΔCt)^{27 (link)}. A dissociation step was performed in samples with SybrGreen primers to test the specificity of the PCR reaction.

The Differential Scanning Fluorimetry (DSF) was accomplished according to Niesen et al. (2007) (link) and Waters et al. (2015b) (link) with slight modifications. An Applied Biosystems 7500 Fast real-time PCR (Thermo Fischer Scientific, United States) was used to follow protein unfolding by monitoring the fluorescence of SYPRO Tangerine (Thermo Fischer Scientific, United States). Protein samples at 0.4 μg/μL (20 μM) in 100 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, and 5% glycerol were screened in the presence and absence of different concentrations of (+)5DS, (-)5DS and DMSO. All reactions contained 5 × SYPRO Tangerine. Aliquots (10 μL) in four replicates were transferred to a 96-well PCR plate and scanned at a ramp rate of 1°C /min from 20 to 80°C. Curve fitting and melting temperatures were calculated using SimpleDSFViewer (Sun et al., 2015 ).

The presence of wild-type H5N1 virus in the chicken swabs at 3 days, 10 days after co-localization, and 3 days, 10 days post-challenge were determined by real-time RT-PCR.Total RNAs were extracted from the chicken swab using an RNA extraction kit (Viral Gene-spin TM Viral DNA/RNA Extraction Kit, iNtRON) according to the manufacturer’s instruction. Next, RNA was subjected to a Onestep RT-PCR kit (Qiagen) with primers and probe targeting the H5 gene (Spackman et al. 2002 (link)). PCR reactions were conducted as follows: reverse transcription at 50 °C for 20 min, denaturation at 95 °C for 5 min, amplification for 45 cycles at 95 °C for 15 s and 60 °C for 30 s. Real-time RT-PCR was carried out on Applied Biosystems 7500 Fast Real-Time PCR (Thermo Fisher). Cycle threshold (Ct) was determined using the 7500 Fast software (Thermo Fisher). Positive samples were detected if Ct was ≤ 35. It was acceptable when the Ct of the positive control was equal to Ct ± 2 of control that was previously known, and the Ct of negative control was 0.