This procedure was performed as previously described (8 (link)). In brief, quantitative PCR (qPCR) was performed to amplify the synthesized cDNA using the RealMaster Mix Reagent (SYBR Green; FP202; Tiangen Biotech Co., Ltd., Beijing, China). An iCycler iQTM Optical Module (Beckman Coulter, Fullerton, CA, USA) was used for RT-qPCR under the following conditions: One cycle at 95°C for 30 sec, 40 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a melt curve between 55 and 95°C in 0.5°C-increments and 10-sec intervals. All the tests were performed in triplicate and the primers used are shown in Table I .
Icycler iqtm optical module
Manufactured by Beckman Coulter
Sourced in United States
The iCycler iQ Optical Module is a component of the iCycler Real-Time PCR Detection System from Beckman Coulter. It is designed to detect and measure the fluorescence signals from real-time PCR reactions. The module is compatible with the iCycler thermal cycler and provides the necessary optics for fluorescence detection during the PCR process.
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3 protocols using icycler iqtm optical module
1
Quantitative PCR Amplification Protocol
2
Quantitative PCR Analysis of Pam3Cys Response
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At 4 h post-stimulation, total RNA was isolated using an RNeasy mini kit (Qiagen, Dusseldorf, Germany) from the Pam3Cys-treated and untreated groups. cDNA was synthesized using the ReverTra Ace quantitative polymerase chain reaction (qPCR) kit (FSQ-101; Toyobo, Kagoshima, Japan). The reverse transcription conditions were 65°C for 5 min, followed by 37°C for 15 min and 98°C for 5 min.
qPCR was performed using RealMaster Mix (SYBR Green; FP202; Tiangen, Beijing, China). The qPCR was performed in an iCycler iQTM Optical Module (Beckman Coulter, Fullerton, CA, USA) under the following conditions: One cycle at 95°C for 30 sec, then 40 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a melt curve from 55 to 95°C in 0.5°C increments and 10-sec intervals. The primers used are listed inTable I . All tests were conducted three times.
qPCR was performed using RealMaster Mix (SYBR Green; FP202; Tiangen, Beijing, China). The qPCR was performed in an iCycler iQTM Optical Module (Beckman Coulter, Fullerton, CA, USA) under the following conditions: One cycle at 95°C for 30 sec, then 40 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a melt curve from 55 to 95°C in 0.5°C increments and 10-sec intervals. The primers used are listed in
3
Quantitative Real-Time PCR Analysis of Gene Expression
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After resuscitation, the H1299, OE, and NC groups were cultured according to ATCC protocol for several days. Total RNA were then isolated using an RNeasy mini kit (Qiagen, Dusseldorf, Germany), from each group on the first, third, fifth, and seventh days, separately. A ReverTra Ace qPCR kit (FSQ −101; Toyobo, Kagoshima, Japan) was used to synthesize cDNA. The conditions for reverse transcription were 65°C for five minutes, 37°C for 15 minutes, then 98°C for five minutes.
All of the PCR primers used were designed according to data provided by GenBank (Table1 ). RealMaster Mix (SYBR Green; FP202; Tiangen, Beijing, China) was used to perform qPCR. All procedures were fulfilled in an iCycler iQTM Optical Module (Beckman Coulter, Fullerton, CA, USA) under the following conditions: initial denaturation at 95°C for 30 seconds, followed by 40 cycles at 95°C for 30 seconds, then 58°C for 30 seconds and 72°C for 30 seconds, followed by a melt curve from 55 to 95°C in 0.5°C increments and 10 second intervals. All tests were repeated three times.
All of the PCR primers used were designed according to data provided by GenBank (Table
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