Abi 7500 rt pcr system

Reverse transcription using 2 μg of each purified RNA sample was done using a Prime Script TMRT Reagent Kit with gDNA Eraser (TaKaRa, China) in accordance with the provided instructions. qRT-PCR was performed on the ABI 7500 RT-PCR system (Applied Biosystems, United States). The composition of the mix was: 10 μL 2× SYBR Premix Ex Taq, 0.4 μL 50× ROX Reference Dye II, 0.4 μM of each primer, and 5 μL of the cDNA template (50-fold dilution) in a net volume of 20 μL. The conditions were: 95°C for 30 s; 95°C for 5 s, 58°C for 15 s, and 72°C for 34 s for 40 cycles. The 2^{[−DeltaDeltaC(T)]} approach was employed to estimate the expression levels of target gene(s) relative (Livak and Schmittgen, 2001 (link)). Ubiquitin was used as an internal reference. Table 1 presents the primers in this study.

We used Trizol reagent to isolate RNA, by following the exact instructions provided by the vendor. The qRT-PCR reactions were performed on an ABI 7500 RT-PCR system (Applied Biosystems). We used primers and detection reagents purchased from Qiagen (China) to detect miR-21. Only RNAse-free water was used throughout the assays.

Total RNA was purified from cells using TRIzol reagent or the Qiagen RNeasy kit and was reverse-transcribed with random primers and RNAseOut according to the manufacturer’s protocol. Gene expression was quantified by real-time quantitative PCR (qPCR) using primers specific for the selected viral or human genes. DNA samples were collected using Qiagen DNeasy Blood and Tissue Kit and were quantified by qPCR with primers specific for viral ICP4 or ICP0 genes or the human 18S ribosomal RNA gene. The qPCR was performed on an ABI 7500 RT-PCR system (Applied Biosystems) using SYBR Green master mix; relative DNA or RNA levels were analyzed by the 2^–ΔΔCt method.

Total RNA extraction and reverse transcription to cDNA were performed according to the manufacturer’s instructions. Quantitative PCR was performed using an ABI 7500 RT PCR system (Applied Biosystems, Germany). For macrophages, the relative gene expression levels of cd11c, inos, ptgs2, cd206, and pparγ were normalized to GAPDH. For PAO1 strain, the relative gene expression level of ndk was normalized to gyrB, rpoD, or rplU gene. The primers used in this study were provided in Supplementary Table S1.

Total RNA was isolated using TRIzol reagent (Invitrogen). For qRT-PCR, RNA (1μg) was used for cDNA synthesis by using a Reverse transcription kit (Takara, Dalian, China) according to the manufacturer's instructions. qRT-PCR analyses were carried out with SYBR Premix Ex Taq (Takara, Dalian China) in the ABI 7500 RT-PCR system (Applied Biosystems, Foster City, USA). Thermocycling parameters: 95°C for 20s followed by 45 cycles of 95°C for 10s and 60°C for 30s. The data was converted to fold change normalized to the expression level of GAPDH, which served as an endogenous control. For PHB mRNA stability analysis by qRT-PCR, 18s ribosomal RNA, a product of RNA polymerase I, was used as an internal control.

Cells collected and the total RNA samples extracted were from CCA cell lines using a Total RNA TriPure Isolation Reagent Kit (BioTeke, Beijing, China) according to the manufacturer’s protocol. RNA was synthesized to first-strand cDNA using the HiscriptII Q RT SuperMix for qPCR with gDNA wiper (R223-01; Vazyme Biotech, Nanjing, China). The reactions were performed for 15 min at 42 °C in a TRIO Thermoblock (Biometra, Goettingen, Germany). Quantitative real-time PCR (qRT-PCR) was performed by an AceQ qPCR SYBR Green Master Mix Kit (Q131-02; Vazyme Biotech, Nanjing, China) in an ABI 7500 RT-PCR system (Applied Biosystems, Foster City, CA, USA) using the following parameters: 1 cycle of pre-denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and 70 °C for 10 s. At this point, mRNA expression was normalized to GAPDH and the relative fold changes were calculated using the 2^−ΔΔCt method. Each sample was run independently in triplicate. The primers for the ARHGAP21 and GAPDH sequences are listed in Table 1.