For APPV and Sendai genome detection, a one-step qRT-PCR format was applied. The 4x TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher Scientific; cat. no. 4444436) was used according to the manufacturer’s recommendations. Briefly, 2 µL of isolated RNA was amplified with 2.5 µL of the 4x TaqMan® Fast Virus 1-Step Master Mix, with 0.4 µM forward and reverse primers and 0.1 µM probe in an end volume of 10 µL. Measurements and analysis were performed using the ABI 7500 Fast Real-Time PCR System instrument and software package (Applied Biosystems, Foster City, CA, USA) using the following thermal profile: 50 °C for 5 min., 95 °C for 20 s, and 45 cycles of 95 °C for 3 s, followed by 60 °C for 30 s. Positive and negative controls where included in every run. For the screening of Linda virus, a SYBR Green one-step qRT-PCR format was used as, based on the limited sequence information available, designing a probe was not feasible. We used the Qiagen QuantiTect SYBR Green OneStep RT-PCR Kit (Qiagen, Hombrechtikon, Switzerland; cat. no. 204243), according to the manufacturer’s recommendations.
Quantitect sybr green onestep rt pcr kit
Manufactured by Qiagen
Sourced in Germany
The QuantiTect SYBR Green OneStep RT-PCR Kit is a laboratory reagent designed for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. It provides a one-step solution for the detection and quantification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Biophotometer, by Eppendorf (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
M mulv reverse transcriptase, by New England Biolabs (1 mentions)
Turbo dna free, by Thermo Fisher Scientific (1 mentions)
Mastercycler ep realplex2 real time pcr system, by Eppendorf (1 mentions)
Steponeplus cycler, by Thermo Fisher Scientific (1 mentions)
Lipid tissue midi kit, by Qiagen (1 mentions)
7500 fast real time pcr, by Thermo Fisher Scientific (1 mentions)
9 protocols using quantitect sybr green onestep rt pcr kit
1
One-step qRT-PCR for APPV, Sendai, and Linda Viruses
2
Quantitative RNA Expression Analysis
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Total RNA was extracted from cells or lungs of rats using Trizol (Invitrogen) according to the manufacturer's instructions. Two micrograms of total RNA were used to synthesize first-strand cDNA with M-MuLV reverse transcriptase (New England BioLabs) using random primers. Real-time PCR was performed using the BioRad iCycler iQ5 Real-Time PCR Detection System with the Quantitect SYBR Green One-Step RT-PCR Kit (QIAGEN). Fluorescence curves were analyzed with iCycler iQ5 Optical System Software (Version 2.0).
3
Gene Expression Analysis in Poultry
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Using quantitative real-time PCR (RT-qPCR), the genes regulating the expression of cytokines (INF-γ, IL-1β, and IL-4); TLR-4; IgA; and Muc-2 in the four experimental groups were analyzed. The relative standard curve of gene expression method was used for quantification of the mRNA expression levels. The gene primers were selected (Table 1 ), and total mRNA was extracted from cecal tissues. Consequently, mRNA was eluted in RNase-free water and stored at -80 °C until use. The purity of the eluted mRNA was evaluated (A260/A280 ratio, Bio-Photometer; Eppendorf, Hamburg, Germany). For RT-qPCR and the mRNA expression rates of avian cytokines, the amplification cycling condition of each tested gene was done according to the listed references [33 –35 ] (Table 1 ). The QuantiTect SYBR Green one-step RT-PCR kit (Qiagen) was used according to the instructions of the manufacturer using Mx3000P real-time qPCR equipment (Stratagene, La Jolla, CA) with the cycling conditions of each primer [36 (link)] (Table 1 ). The abundance of each target mRNA was assessed by a 2 − ∆∆CT comparative method [36 (link)] with normalization against β-actin in the following formula: − ∆∆CT (sample-control) = (CT of target gene- CT of β-actin gene) sample − (CT of target gene − CT of β-actin gene) control. Results were expressed as a fold change.![]()
Primers used in real-time quantitative PCR
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated by acid phenol/chloroform extraction as described previously
[11 (link)]. The yield and the purity of RNA were determined by measuring absorption at 260 nm. Total mRNA samples were treated with TURBO DNA-free (Applied Biosystems, Darmstadt, Germany) to remove remaining traces of genomic DNA as described by the manufacturer’s recommendation. SYBR-green based qRT-PCR was performed with 5 ng RNA template and 100 μM primer with QuantiTect SYBR Green one-step RT-PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The thermocycler program comprised an initial step of 95°C for 15 min followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s. Reactions were performed with biological triplicates in a Mastercycler ep realplex2 real-time PCR system (Eppendorf, Hamburg, Germany) as described by the manufacturer using their universal program. Reactions with no addition of reverse transcriptase served as negative control and proved the absence of DNA contamination. Specificity of amplification was assessed by analyzing the melting curve of the amplification product. Primers to amplify lscB were used for constructs lscB and lscAUpB while primers to amplify lscA were used for constructs lscA, lscBUpNA and lscBUpA. All the results were normalized to amplification of the cDNA of gyrA (PSPPH3667) as described previously
[43 (link)].
[11 (link)]. The yield and the purity of RNA were determined by measuring absorption at 260 nm. Total mRNA samples were treated with TURBO DNA-free (Applied Biosystems, Darmstadt, Germany) to remove remaining traces of genomic DNA as described by the manufacturer’s recommendation. SYBR-green based qRT-PCR was performed with 5 ng RNA template and 100 μM primer with QuantiTect SYBR Green one-step RT-PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The thermocycler program comprised an initial step of 95°C for 15 min followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s. Reactions were performed with biological triplicates in a Mastercycler ep realplex2 real-time PCR system (Eppendorf, Hamburg, Germany) as described by the manufacturer using their universal program. Reactions with no addition of reverse transcriptase served as negative control and proved the absence of DNA contamination. Specificity of amplification was assessed by analyzing the melting curve of the amplification product. Primers to amplify lscB were used for constructs lscB and lscAUpB while primers to amplify lscA were used for constructs lscA, lscBUpNA and lscBUpA. All the results were normalized to amplification of the cDNA of gyrA (PSPPH3667) as described previously
[43 (link)].
5
RNA Extraction and Real-Time RT-PCR Analysis
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RNA was prepared from tissue stored in RNAlater at −80°C following the protocol of the manufacturer (lipid tissue midi kit; Qiagen). Concentration and purity of samples were assessed using a NanoDrop spectrophotometer (Pierce, Wilmington, DE). RNA was diluted to 50 ng/μL and applied to Real-Time RT-PCR using the QuantiTect SYBR Green One Step RT-PCR Kit (Qiagen) and the following primers: abcb1a QT01753416, abcb1b QT00140945, gapdh QT00309099 (all Qiagen). RT-PCRs were performed on a StepOnePlus Cycler (Applied Biosystems, Darmstadt, Germany) with 100 ng RNA per reaction. Relative quantities were calculated using appropriate standard curves.
6
Quantitative Gene Expression Analysis in AoSMCs
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QuantiTect Primer Assays determined gene expression for prostaglandin‐endoperoxide synthase 2, also known as cyclooxygenase‐2 (PTGS2; QT00040586), CNN1 (QT00067718), ERK1 (QT00065933), ERK2 (QT02589321), AKT1 (QT00085379), Nuclear Factor Kappa B Subunit 1 (NFKB1; QT00063791), MMP2 (QT00088396), and MMP9 (QT00040040) in human AoSMC using quantitative real time polymerase chain reaction as previously described.15 The relative expression of these genes in experimental and control samples was calculated by using the concentration‐Ct‐standard curve method and normalized using the average expression of GAPDH (human GAPDH, QT00079247) for each sample using the Rotor‐Gene Q operating software version 2.0.24. The QuantiTect SYBR Green one‐step RT‐PCR Kit (Qiagen) was used according to the manufacturer's instructions with 40 ng of total RNA as template. All reactions were independently repeated in duplicate.
7
Quantitative Real-Time PCR Analysis of Genes
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QuantiTect® Primer Assays were used to determine gene expression for Par-1 (QT00119812), Par-2 (QT02255330), Mmp2 (QT00116116), and Mmp9 (QT00108815) in mouse aortic tissue, and, SMAD2 (QT00004207), SMAD3 (QT00008729), MMP2 (QT00088396) and MMP9 (QT00040040) in human VSMCs using quantitative real time (qPCR) as previously described48 (link). The relative expression of these genes in experimental and control samples was calculated by using the concentration-Ct-standard curve method and normalized using the average expression of glyceraldehyde-3-phosphate dehydrogenase (mouse Gapdh, QT01658692; human GAPDH, QT00079247) for each sample using the Rotor-Gene Q operating software version 2.0.24. The QuantiTect SYBR® Green one-step RT-PCR Kit (Qiagen) was used according to the manufacturer’s instructions with 40ng of total RNA as template. All reactions were independently repeated in duplicate to ensure the reproducibility of the results. For mouse studies, six SRAs were randomly selected from the vehicle, fondaparinux, and DE groups using an online random number generator (https://www.random.org/ ).
8
Quantitative RT-PCR Analysis Protocol
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Primer design was performed using NCBI primer-BLAST. To avoid amplification of non-specific DNA, when applicable, primers were required to span an exon-exon junction and the primer pair was to be separated by at least one intron on the corresponding genomic DNA (Supplemental Table 2 ). Total RNA was isolated using the RNeasy Mini kit (Qiagen, 74104). Quantitative RT.PCR analysis was performed on a 7900HT real time thermocycler using the QuantiTect SYBR Green one-step RT.PCR kit (Qiagen, 204243). The SDS software (ABI, version 2.4) was used to analyze the data and additional analysis was performed on Microsoft Excel. Relative quantification was performed using the ΔΔCt method and statistical significance was determined using the T-Test. The final reaction product was run on an agarose gel to determine whether the size of the amplimer detected and quantified was the expected.
9
Quantitative Analysis of Cytokine Gene Expression
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Total RNA from the tissue samples of the distal jejunum (20 mg) was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted into 50 μL of Rnase free water and stored at −80 °C. The yield of RNA was determined by spectrophotometry (BioPhotometer, Eppendorf, Hamburg, Germany). Cytokine gene expression (IL-6, IL-1β, IFN-γ and IL-10) were evaluated as previously described by Reid et al. [18 (link)]. The mRNA quantification of cytokines was determined by qRT-PCR using QuantiTect™ SYBR® Green one-step RT-PCR Kit (QIAGEN, Hilden, Germany). The PCR amplification was performed using 7500-Fast Real-time PCR (Applied Biosystems, CA, USA) [18 (link)].The threshold cycle values (Ct) were t normalized to the reference gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)). The average ∆Ct of the control samples was used to calculate the target gene expression according to the 2−∆∆Ct method [19 (link)]. This relative quantification related the PCR signal of the target transcript gene in a treatment group to the average signal of untreated control. Duplicate samples were used.
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