Total RNA of 1×106 cells of MICAL2-knockdown HCT116 or MICAL2-knockdown SW480 was extracted using Trizol reagent (Life Technologies) following the manufacturer's instructions. 1 μg DNase-treated RNA was reverse transcribed using Revert AidTM First-Strand cDNA Synthesis Kit (MBI Fermentas, USA) according to the manufacturer's instructions. The threshold cycle (Ct) value of each sample was determined using Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) in ABI 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA). Sequences of primers used are shown in Table S3 . Relative mRNA expression of each target gene was normalized to the expression of the housekeeping gene GAPDH. Relative mRNA level was calculated as two power values of ΔCt (Ct value of GAPDH Ct of target gene).
Platinum sybr green qpcr supermix udg with rox
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Japan
The Platinum SYBR Green qPCR SuperMix-UDG with ROX is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains SYBR Green I dye for detection, the UDG enzyme for carry-over prevention, and the ROX passive reference dye.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (7 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (7 mentions)
Quantitect reverse transcription kit, by Qiagen (7 mentions)
Rneasy kit, by Qiagen (7 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (6 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (5 mentions)
Rq1 rnase free dnase kit, by Promega (5 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (3 mentions)
Thermoscript rt pcr system, by Thermo Fisher Scientific (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Rneasy plant mini kit, by Qiagen (3 mentions)
Superscript 2, by Thermo Fisher Scientific (3 mentions)
Oligo dt and superscript 2, by Thermo Fisher Scientific (3 mentions)
Revertaidtm first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
83 protocols using platinum sybr green qpcr supermix udg with rox
1
Quantifying MICAL2 Gene Expression in Colorectal Cancer
2
qRT-PCR analysis of cadherin expression
3
ChIP Assay for Plasma Cell Epigenetics
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ChIP was performed on DMSO or UNC1999 treated cell lines and CD138+ purified plasma cells using a modified version of the OneDay ChIP kit (Diagenode, Liège, Belgium) as previously described [18 (link)]. Chromatin was crosslinked with 1% formaldehyde for 10 minutes at room temperature, followed by 5 minutes treatment with 1 M glycine to stop crosslinking. Chromatin was sonicated (30sec ON/30 sec OFF) for 6 cycles of 5 minutes each at ultrasonic wave output power 320 W in Bioruptor® (Diagenode, Liège, Belgium). Cells were collected and lysed on ice in RIPA extraction buffer with a cocktail of protease inhibitors. Antibodies used are listed in Supplementary Table 4 . Precipitated DNA was analyzed by Real Time-qPCR using Platinum® SYBR® Green qPCR SuperMix UDG with Rox (Invitrogen, Carlsbad, CA) and 0.25 mM of each forward and reverse primers. Primer sequences used in this study were, miR-125a: forward primer: 5´-CCCTTCCTCCAGAGCATGAC-3` and reverse primer: 5´-CCATCGTGTGGGTCTCAAGG-3´; miR-320c2: forward primer: 5´-GTTGAGGAGCACTGGGTATGT-3´and reverse primer: 5´-CTTACCCTCTCAACCCA GCTT-3´. The PCR conditions were: 95°C for 2 min followed by 40 cycles of 95°C for 0:30 min and 60°C for 1 min. The run and analysis were performed using Mx3005P instrument and software (Stratagene).
4
Reverse Transcription and Real-Time PCR Analysis
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Total RNA was isolated by using the Quick RNA Miniprep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. RNA was reverse transcribed with Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and with polyT primers (Thermo Scientific/Fermentas, Waltham, MA, USA). Quantitative amplification was conducted on a PCR performed on a QuantStudio12K Flex Real Time PCR System (Life Technologies, Carlsbad, CA, USA) with gene-specific primers and Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen). A newly synthesized transcript analysis was performed as previously described [20 (link)]. RNA was reverse transcribed and analyzed by real-time PCR.
5
Quantification of CaN and NFATc2 Expression
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Total RNA was extracted from the pulmonary arteries using an E.Z.N.A. Total RNA I kit (Omega Biotek, Inc., Norcross, GA, USA), which included tissue lysing and homogenization, according to the manufacturers instructions. Subsequently, the total RNA was reverse transcribed into cDNA using a Moloney Murine Leukemia Virus Reverse Transcriptase kit (Invitrogen Life Technologies). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses for CaN and NFATc2 were performed on an 7300 Real-Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA) using Platinum® SYBR® Green qPCR SuperMix-UDG with ROX (Invitrogen Life Technologies). The primer sequences were as follows: GAPDH, forward 5′-CCA TTC TTC CAC CTT TGA TGCT-3′, reverse 5′-TGT TGC TGT AGC CAT ATT CAT TGT-3′; CaN, forward 5′-CAG AGG GTG CTT CGA TTCTC-3′, reverse 5′-CCC CTA AGA AGA GGT AGC GA-3′; and NFATc2, forward 5′-CAG CAG ATT TGG GAG ATG GAA G-3′, and reverse 5′-GAC TGG GTG GTA AGT AAA GTG C-3′. PCR cycling conditions were as follow: 95°C for 2 min, and 95°C for 15 sec, and 60°C for 30 sec, for 40 cycles. The relative expression levels were quantified by the 2−ΔΔCt method.
6
Quantifying RNA Expression in Hepatoma Cells
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Total RNA was extracted from HepG2, HepG2 SR, Huh7, and Huh7 SR cells using an HP Total RNA Kit (Omega Biotech, Stamford, CT, USA) according to the manufacturer’s instructions. The concentration of the RNA samples was measured on a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). The synthesis of cDNA was carried out with the enzyme reverse transcriptase using an M-MLV First Strand kit (Life Technologies, Gaithersburg, MD, USA). Polymerase chain reaction (PCR) were performed using the Platinum®SYBR®Green qPCR supermix-UDG with ROX (Invitrogen) on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The cycling conditions were the following: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles, each consisting of 95°C for 15 s and 60°C for 1 min. The relative amounts of the Ct values of the gene mRNA- and microRNA-expression levels were normalized to those of β-actin mRNA and U6, respectively, which served as internal controls. The fold change was calculated using the 2–ΔΔCt method.
7
Gene Expression Analysis in Macrophages
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Two-step real-time quantitative PCR (qPCR) was performed to characterize the expression of selected genes in macrophages. Total RNA isolation was performed with TRIzol reagent (Invitrogen). The Quantitect reverse transcription (RT) kit (Qiagen, Tokyo, Japan) was used to obtain the complementary DNAs following the manufacturer’s recommendations. The Platinum SYBR green qPCR SuperMix UDG with ROX (Invitrogen) and the 7300 real-time PCR system (Applied Biosystems, Warrington, UK) were used for qPCR. Table S1 show the sequence of primers used in this work. The PCR cycling conditions were described previously [4 (link)]. The reaction mixtures contained 5 μL of sample cDNA and 15 μL of master mix, including primers. β-actin expression was used to normalize cDNA levels in the samples.
8
Porcine Monocyte-Derived Dendritic Cell Generation
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Porcine monocyte-derived dendritic cells (MoDCs) were generated as previously described [32 (link)]. Briefly, porcine monocytes were obtained from freshly collected porcine peripheral blood by a density gradient centrifugal method (1800 rpm, 20 min, 20 °C) with Lympholyte-mammal (Cedarlane, Hornby, ON, Canada). Cells were suspended in RPMI and plated (1 × 107 cells/mL per well) into 12-well plates (Corning, Brumath, France) in RPMI-1640 supplemented with 2% FCS, 1% Streptomycin/Penicillin, and incubated two hours. Non-adherent cells were removed and remaining cells were incubated with RPMI supplemented with 10 μL/well of porcine GM-CSF (20 ng/mL) and 10 μL/well of porcine IL-4 (20 ng/mL). Fresh medium was renewed every 2 days. After 5 days of culture, cells were incubated with RPMI medium supplemented with GM-CSF, IL-4, and 5 μL/well of LPS (200 μg/mL) in order to induce differentiation of MoDC. The qRT-PCR was performed to quantify the expression of PGLYRPs mRNAs in porcine MoDCs. qRT-PCR was carried out with using Platinum SYBR Green qPCR SuperMix UDG with ROX (Invitrogen, Carlsbad, CA, USA). The primers used in this study are listed in Supplementary Table S2 .
9
Quantitative PCR Analysis of RNA Samples
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The samples were harvested in TRIzol KS reagent (Life Technologies) and the total RNA was extracted by phase‐separation with chloroform followed by RNA isolation and purification using the RNeasy kit (Qiagen) according to the manufacturer instructions. The total RNA content of each sample was quantified using NanoDrop system (Thermofisher Scientific, USA), and the cDNA was synthesized using the SuperScript III First‐Strand Synthesis System kit (Thermofisher). The RT‐qPCR analysis (n = 3; duplicate measurements) was performed on the ViiA 7 Real‐Time PCR System (Applied Biosystems, USA) using Platinum SYBR Green qPCR Supermix‐UDG with ROX (Invitrogen, CA, USA) and the primer sequences were listed in Table S12 . Gene expression (cycle threshold, CT) values were normalized to the housekeeping gene GAPDH (Glyceraldehyde‐3‐phosphate dehydrogenase) and the delta CT (dCT) values were calculated.
10
RNA Extraction and qRT-PCR for Henipavirus Detection
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After a first step using RLT buffer (Qiagen, Venlo, Netherland) supplemented with 0.1% beta-mercaptoethanol, RNA was purified from tissues in accordance with the manufacturer’s instructions (Kit NucleoSpin RNA Macherey Nagel). Purified RNA was then treated with Turbo DNaseI (Ambion, Life Technologies, Delhi, India) and subjected to reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad, Marnes La Coguette, France).
RNA from oropharyngeal swabs was extracted and purified using the QIAamp viral RNA Mini Spin kit (Qiagen). The qRT-PCR reaction was conducted on 10 ng of complementary DNA, using the PlatinumSYBRGreen qPCR SuperMix-UDG with ROX (Invitrogen) was run on the Step One Plus Real Time PCR System (Applied Biosystems, Waltham, MA, USA). Sequences of primers that target HeV NP gene and GAPDH gene were as previously described.37 (link) All samples were run in duplicate, and results were analysed using the ABI StepOne software v2.1 (Applied Biosystems).
RNA from oropharyngeal swabs was extracted and purified using the QIAamp viral RNA Mini Spin kit (Qiagen). The qRT-PCR reaction was conducted on 10 ng of complementary DNA, using the PlatinumSYBRGreen qPCR SuperMix-UDG with ROX (Invitrogen) was run on the Step One Plus Real Time PCR System (Applied Biosystems, Waltham, MA, USA). Sequences of primers that target HeV NP gene and GAPDH gene were as previously described.37 (link) All samples were run in duplicate, and results were analysed using the ABI StepOne software v2.1 (Applied Biosystems).
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