7500 real time pcr system instrument

Unless otherwise specified, the melting analysis was carried out in 96-well plate with 1.2 μl of second-round PCR reaction product, 2 μl of IQ SYBR Green supermix or Precision Melt Supermix containing EvaGreen Dye (Bio-Rad) and 0.16 μl of ROX Passive Reference Dye (Bio-Rad) in a 8 μl reaction mixture using a 7500 real Time PCR System instrument (Applied Biosystems). The following melt curve run method was used: 95°C for 15 s, 60°C for 1 min followed by an increase to 95°C with a ramp rate of 1% and continuous data acquisition (40 min long). When specified, melt profiling was performed using either CFX96 Touch or CFX384 Touch Real time PCR detection system instruments (Bio-Rad) with the same melting reaction mixture as given above except that no ROX Passive Reference Dye was added. Melting run method was as follows: 95°C for 15 s, 60°C for 1 min followed by an increase to 95°C with an increment of 0.5°C and reading after 2 s (25 and 35 min long for 96 and 384-well instruments respectively). Melting profile was analyzed to isolate the positive reactions with the onboard software using the methods described in the text.

Whole blood was collected into PAXgene blood tubes (Qiagen, Redwood City, CA) and stored at -80°C until use. Total RNA was isolated with on-column DNase I digestion using the PAXgene Blood miRNA kit (Qiagen). Total RNA was further isolated from liver tissue using the RNeasy Mini kit (Qiagen) with on-column DNase I digestion using RNase-free DNase (Qiagen). Following reverse transcription of mRNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) using oligo(dT), complementary (c) DNA samples were amplified on a 7500 Real Time PCR System instrument (Applied Biosystems) using TaqMan or SYBER Green Gene Expression Master Mix (Applied Biosystems). Target genes investigated (S1 Table) included markers for: (i) type I IFNs and ISGs; (ii) NK-cells; (iii) APCs; (iv) Th-cells; (v) CTLs; and (vi) T_regs. The woodchuck-specific primers and probes are presented in S2 Table. 18S rRNA expression was used to normalize target gene expression. Transcript levels of target genes were calculated as a fold-change relative to the pre-inoculation (baseline) level in blood and liver at week -2 (or at week 0 prior to inoculation in blood of woodchuck F7394 and woodchucks M7392 and M7249 for selected genes) using the formula 2^-ΔΔCt. A fold-change of ≥2.1 from this baseline was considered a positive result for the presence of increased target gene expression.

Commercially available TaqMan real-time PCR kits were used to detect NG and CT in the clinical specimens. All the procedures were performed according to the directions and recommendations in the manufacturer’s protocols (Neisseria gonorrhoea and Chlamydia trachomatis standard kits; Primer Design Ltd., Millbrook Technology Campus, Southampton, UK). The amplification was performed using the TaqMan Gene Expression Master Mix Reagents (Applied Biosystems, Foster City, CA, USA) in a 7500 Real-Time PCR System instrument (Applied Biosystems, USA).

Biomass samples of the wild-type and the recombinant strains cultured in SFM for 4 d were taken out, and total RNA was separated using Total RNA Extractor (Shanghai Sangon Biotech Co., Ltd.) according to the instructions. The RNA concentration and purity were determined by measuring the ratio of OD₂₆₀ to OD₂₈₀. The 7500 Real-Time PCR system instrument (Applied Biosystems, USA) was used to determine the transcription level of the sample. Prime Script^TM RT Reagent Kit (Takara) were used for DNase treatment and cDNA synthesis according to the instructions. SYB^® Permix Ex Tag^TM GC (Takara) was used for qRT-PCR amplification. The primer pairs used in qRT-PCR were listed in Additional file 1: Table S2, and the 16S rRNA gene was used as an internal control to quantify the relative expression level of the samples.

Total RNA from NSCLC, NMLT, and cell line sections was isolated using the RNeasy Mini Kit (catalogue no. 74104, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To remove genomic DNA, the samples were digested using the RNase-Free DNase Set (catalogue no. 79254, Qiagen, Hilden, Germany). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription kit with the RNase inhibitor (catalogue no. 4374966, Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The qPCR was performed using the 7500 Real-Time PCR System instrument and 7500 software v2.0.6 (Applied Biosystems, Foster City, CA, USA). The following Taqman probe and primer sets were used in the reactions: ZYX (Hs00170299_m1, Applied Biosystems, Foster City, CA, USA) and ACTB (Hs99999903_m1, Applied Biosystems, Foster City, CA, USA). Real-time PCR reaction conditions were as follows: polymerase activation at 50 °C for 2 min, initial denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 sec, annealing and extension at 60 °C for 1 min for 45 cycles. β-actin (ACTB) was used as the reference gene. Changes in gene expression were determined using the ΔΔCt method [28 (link)]. Reactions were performed in triplicate.

Total DNA from larvae was extracted using phenol/chloroform precipitation. The amount of mtDNA was assessed by the ratio of mtDNA to nuclear DNA (nDNA) copy number determined by quantitative real time amplification of the mitochondrial 16S gene and the nuclear Rpl32 gene. Primers used in this work (16S F and R; Rpl32 F and R) were those reported previously (29 (link)). We generated two gene-specific calibration curves with six 10-fold serial dilutions (100–10,000,000 copies) of plasmids containing the cloned target sequences (Invitrogen). Concentration of plasmid stock solutions was assessed with an ND-1000 spectrophotometer (NanoDrop), and the plasmid copy number of dilutions was calculated using Avogadro's number. Reactions were performed in triplicate using SYBR Green chemistry according to the manufacturer's recommendations (GoTaq qPCR Master Mix, Promega) in a 7500 Real Time PCR System instrument (Invitrogen). Data were normalized to the ratio of mtDNA/nDNA copy number in controls (arbitrary set to 100%) (31 (link), 32 (link)).