The qPCR reaction system consisted of 5 µl SYBR Green Premix Taq (cat. no. RN04006M; Monad Biotech Co., Ltd.), 1 µl cDNA, 0.3 µl forward primer (10 µM), 0.3 µl reverse primer (10 µM) and 3.4 µl H2O. The thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec, 60˚C for 30 sec and 72˚C for 15 sec. Dissolution curve was produced using the standard dissolution curve program (Agilent Aria Software; version 1.5; Agilent Technologies, Inc.). The PCR reaction was carried out on an ABI 7500 quantitative PCR instrument (Thermo Fisher Scientific, Inc.). The relative mRNA expression data were calculated using the 2-ΔΔCq method (20 (link)). The relative expression value of a gene was normalized against the expression of Actb from control group (WNS) mRNA. The Data was analyzed using the SPSS 22.0 software (IBM Corp). Primer sequences are presented in Table I .
Agilent aria software
Manufactured by Agilent Technologies
Sourced in United States
Agilent Aria software is a data acquisition and analysis platform designed for Agilent's analytical instrumentation. It provides essential functionalities for collecting, processing, and managing data generated by Agilent's analytical equipment.
Automatically generated - may contain errors
Lab products found in correlation
Qiamp viral rna mini kit, by Qiagen (3 mentions)
Ariamx real time pcr system, by Agilent Technologies (3 mentions)
Luna universal one step rt qpcr kit, by New England Biolabs (2 mentions)
Abi7500 quantitative pcr instrument, by Thermo Fisher Scientific (1 mentions)
Spss 22, by IBM (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
4 protocols using agilent aria software
1
qPCR Methodology for Gene Expression Analysis
2
TBEV RNA Quantification from Organ Homogenates
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The total RNA of supernatants of cleared organ homogenates was isolated using QIAmp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Real-time reverse transcriptase-quantitative PCR (RT-qPCR) was performed using Luna® Universal One-Step RT-qPCR Kit (New England BioLabs® GmbH, Frankfurt (Main), Germany) based on the modified protocol by Schwaiger and Cassinotti (43 (link)). For the quantification of TBEV RNA copies, a TBEV RNA standard (kindly provided by Stefanie Becker, Institute for Parasitology and Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany) was used. Nuclease-free water served as a negative control. Real-time RT-qPCR was performed with AriaMx Real-time PCR System with Agilent Aria software (version 1.5, Agilent Technologies, Santa Clara, California, USA). For data evaluation, quantification cycle (Cq) values of duplicates were averaged, and TBEV copies per gram of tissue were calculated. Data were expressed as log10 TBEV copies per gram tissue. In samples with no detectable viral RNA (no Cq value could be measured), calculation to log10 resulted in 1 (100) copy per gram of tissue.
3
TBEV RNA Quantification in Sera and Tissues
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Total RNA was extracted from sera and clarified organ homogenate supernatants using the QIAmp® Viral RNA Mini Kit (Qiagen) following manufacturer’s protocol. Real time reverse transcriptase quantitative PCR (RT-qPCR) was performed with Luna® Universal One-Step RT-qPCR Kit (New England BioLabs® GmbH) based on the modified protocol by Schwaiger and Cassinotti (50 (link)) including a TBEV Neudoerfl RNA standard kindly provided by Stefanie Becker (Institute for Parasitology and Research Center for Emerging Infections and Zoonoses at University of Veterinary Medicine Hannover, Foundation, Hannover, Germany). Real time RT-qPCR was run in duplicates using AriaMx Real-time PCR System (Agilent Technologies) with Agilent Aria software (version 1.5, Agilent Technologies). Absolute copy numbers were calculated based on the standard curve and expressed as TBEV copies/ml or g tissue.
4
TBEV RNA Quantification in Serum and Tissues
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Total RNA of serum (pre-diluted 1:10 in A549 medium) or organ homogenate (free of cell debris) was isolated using QIAmp® Viral RNA Mini Kit (Qiagen) according to manufacturer’s manual. For detection of TBEV RNA, real time quantitative reverse transcription (RT)-PCR using One-Step RT-PCR Kit (Qiagen) was performed based on the protocol established by Schwaiger and Cassinotti (21 (link)) with few modifications. To determine TBEV RNA copies, a dilution row of TBEV Neudoerfl RNA standard was used. The standard was kindly provided by Stefanie Becker (Institute for Parasitology and Research Center for Emerging Infections and Zoonoses at University of Veterinary Medicine Hannover, Foundation). As negative control, AVE buffer instead of sample was used. Real time quantitative RT-PCR was performed in duplicates using AriaMx Real-time PCR System (Agilent Technologies) with Agilent Aria software (version 1.5, Agilent Technologies). Cq values were converted into log10 TBEV copies/ml or gram tissue, respectively, according to the standard curve.
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