saSE tissues were homogenized by TissueLyser II with Qiagen’s stainless beads (5mm). Total RNA was isolated from saSE tissues using the QIAshredder and QIAcube (Qiagen) according to the manufacturer’s protocol. RNA integrity and concentration were assessed using the Agilent 4200 TapeStation and all samples met quality control criteria using RNA ScreenTape (Agilent technologies). 0.7μg RNA from each sample was used to synthesize cDNA using the iScript cDNA synthesis kit (Bio-Rad). Quantitative real time PCR (qRT-PCR) was performed using TaqMan probe single tube assays (Life Technologies) for CD163, CD206, ITGAM and CD14. A StepOnePlus Real-Time PCR System (Applied Biosystems) was used for amplification and detection. Threshold cycle number was determined using Opticon software. Thermal cycling conditions consisted of an initial incubation at 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Product accumulation was measured during the extension phase, and all samples were run in triplicate. The relative mRNA levels of CD163, CD206 and ITGAM were normalized to CD14 and represented as relative mRNA expression determined by ΔΔCT method.
Taqman probe single tube assays
Manufactured by Thermo Fisher Scientific
TaqMan Probe single tube assays are a method for detecting and quantifying specific DNA sequences in real-time PCR. The assays use a fluorogenic probe that consists of an oligonucleotide with a reporter dye at the 5' end and a quencher dye at the 3' end. During PCR amplification, the probe binds to its complementary sequence and the reporter dye is cleaved, resulting in an increase in fluorescence that is detected and measured.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Stainless beads, by Qiagen (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Qiacube, by Qiagen (1 mentions)
Rna screentape, by Agilent Technologies (1 mentions)
Qscript xlt cdna supermix, by Quantabio (1 mentions)
Quick rna microprep kit, by Zymo Research (1 mentions)
Allprep rna protein kit, by Qiagen (1 mentions)
5 protocols using taqman probe single tube assays
1
Macrophage Marker Expression Profiling
2
Quantitative Real-Time PCR Analysis of Inflammatory Genes
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Quantitative real time PCR was performed using TaqMan Probe single tube assays (Life Technologies) for mouse (Ccl2, Cxcl16, Arg1, Il1b, Cd36, and Mmp2) genes. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used for amplification and detection. Threshold cycle number was determined using Opticon software. mRNA levels were normalized to β−actin, which control studies showed is not altered by CDDO-Me treatment, using the equation 2−(Et-Rt), where Rt is the mean cycle threshold for the control gene and Et is the mean threshold for the experimental gene. Thermal cycling conditions for qRT-PCR consisted of an initial incubation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Product accumulation was measured during the extension phase and all samples were run in triplicate12 (link).
3
Quantitative Analysis of Gene Expression
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Total RNA from human and mouse cells was obtained using the miRNeasy Mini Kit (Qiagen) per manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 100 ng total RNA and random hexamers using the SuperScript III First-Strand Synthesis System (Life Technologies). Quantitative real time PCR (qRT-PCR) was performed using TaqMan Probe single tube assays (Life Technologies) for human (CCL18, IL-6, IL-10, VEGF, TNF-α, CCR5, CD163, CLEC10A) and mouse (IL-10, Arg1, Ym1, TNF, CXCL9) genes. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used for amplification and detection. Threshold cycle number was determined using Opticon software. mRNA levels were normalized to β-actin, which control studies showed is not altered by CDDO-Me treatment, using the equation 2-(Et-Rt), where Rt is the mean cycle threshold for the control gene and Et is the mean threshold for the experimental gene. Thermal cycling conditions for qRT-PCR consisted of an initial incubation at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Product accumulation was measured during the extension phase and all samples were run in triplicate.
4
Quantitative RNA Expression Analysis
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Total RNA was isolated using the quick RNA microprep kit (Zymo, Cat. #11-328M) per manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 100 ng total RNA and random hexamers using the qScript™ XLT cDNA SuperMix (QuantaBio, Cat. #955161-100). Quantitative real time PCR (qRT-PCR) was performed using TaqMan Probe single tube assays (Life Technologies, Cat. #4324018) for human CCL2 (Applied Biosystems, Cat. # Hs00234140), IL-6 (Cat. #Hs00985639_m1) and VEGF (Cat. # Hs00900055_m1) genes. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used for amplification and detection. Threshold cycle number was determined using Opticon software. mRNA levels were normalized to β-actin, which control studies showed is not altered by CDDO-Me treatment (11 (link)), using the equation 2-(Et-Rt), where Rt is the mean cycle threshold for the control gene and Et is the mean threshold for the experimental gene. Thermal cycling conditions for qRT-PCR consisted of an initial incubation at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Product accumulation was measured during the extension phase and all samples were run in triplicate.
5
Quantitative PCR Analysis of Cytokine Genes
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