Abi 7500 fast thermal cycler

To determine whether Gdnf and Manf mRNA is expressed in microglial SIM-A9 cells, total RNA was extracted from cells in TRI reagent (T9424, Sigma-Aldrich), digested with DNase I and converted to cDNA using iScript gDNA Clear cDNA Synthesis Kit (172-5034, Bio-Rad Laboratories, Hercules, CA, USA). To check whether target genes were amplified from contaminated genomic DNA, negative control without reverse transcriptase was included. Mouse hypothalamic cDNA was used as a positive control. PCR was performed for 40 cycles at 95 °C for 3 s and 60 °C for 30 s. PCR products along with a 50 bp DNA ladder (10416014, Thermo Fisher Scientific, Waltham, MA, USA) were separated on 3% agarose gel in 1X TAE and visualized under UV light.
Expression levels of mRNA were measured by real-time PCR using the ABI 7500 Fast thermal cycler (Applied Biosystems, Foster City, CA, USA) as described previously [29 (link)]. All primer pairs (Table 1) were designed using the NCBI Primer-Blast tool. Relative mRNA levels were determined using ΔΔCt method by normalizing to hypoxanthine guanine phosphoribosyl transferase (Hprt) mRNA levels. All experiments were performed in triplicates and the coefficient of variation (CV) was less than 5% for each triplicate.

RNA was extracted from 100 μl nasopharyngeal samples collected from experimentally infected horses using the RNAgents Total RNA Isolation System (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer's instructions. One-step real-time RT-PCR was performed using the Light Cycler RNA Amplification kit, SYBR Green I (Roche, Burgess Hill, West Sussex, UK) as previously described.10 (link)
RNA was extracted from 140 μl nasopharyngeal swabs submitted from clinical samples using the QIAamp Viral RNA Mini Kit (Qiagen, Crawley, West Sussex, UK) in accordance with the manufacturer's instructions. One-step real-time RT-PCR using a primer probe-based assay which targets the matrix gene of influenza A virus22 (link) and an AgPath-ID One-Step RT-PCR Kit (Ambion, Austin, TX, USA) on an ABI 7500 Fast thermal cycler (Applied Biosystems, Austin, TX, USA) platform was carried out. Briefly, 5 μl of purified nucleic acid was added to a 20 μl reaction mix containing 25× RT buffer, 80 ng tRNA (Laborchemikalien GmbH, Seelze, Germany), 0·36 μm of each primer, 0·15 μm of probe and 25× RT enzyme. One-step RT-PCR was carried out at 45°C for 10 minutes followed by 95°C for 10 minutes, 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds.

The total RNA from mouse bronchial epithelial cells was isolated using TRIzol^® reagent. Then, the sample was reverse transcribed from RNA to cDNA using a Prime Script RT kit. Polymerase chain reaction (PCR) amplification was performed on the ABI 7500 Fast Thermal Cycler (Applied Biosystems, USA), according to the SYBR Green PCR Kit (Takara Biotechnology Co., Ltd., Dalian, China). PCR cycle was conducted for 30 s at 95°C, followed by 40 cycles at 95°C for 5 s and an annealing/extension step at 60°C for 15 s. The primer was designed by the Shanghai Sango Company (Shanghai, China). The specific primers are shown in Table 1.