RNA was extracted and purified with a glass fiber filter plate (Pall # 5072) and chiotropic salts. Messenger RNA expression levels were quantitated with quantitative reverse transcription PCR on the QuantStudio7 PCR Instrument (Applied Biosystems). Briefly, 10 μl RT-qPCR reactions containing 4 μl of RNA were run with Agpath-ID reagents (Applied Biosystems) and gene specific TaqMan primer probe sets (Integrated DNA Technologies) following the manufacturer's instructions. Total RNA levels, measured with Quant-iT Ribogreen Reagent (Thermo Fisher), were used to normalize the RT-qPCR data.
Taqman primer probe sets
Manufactured by Integrated DNA Technologies
Sourced in United States
TaqMan primer/probe sets are a type of real-time PCR reagent designed for the detection and quantification of specific DNA or RNA sequences. They include a forward primer, a reverse primer, and a fluorescently labeled probe that binds to the target sequence during amplification. The probe releases a fluorescent signal upon cleavage, allowing for real-time monitoring of the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Prepease rna spin kit, by Thermo Fisher Scientific (2 mentions)
Veriquest probe one step qrt pcr master mix, by Thermo Fisher Scientific (2 mentions)
Quant it ribogreen reagent, by Thermo Fisher Scientific (1 mentions)
7900ht apparatus, by Thermo Fisher Scientific (1 mentions)
Sensifast probe lo rox mix, by Meridian Bioscience (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using taqman primer probe sets
1
Quantitative RT-PCR for mRNA Profiling
2
Neuroinflammatory Profile in Virus Infection
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mRNA expression of IL-6, CXCL10, granzyme B, IFN-γ, IL-1β, and IL-10 was determined in RNA extracts from brains 8 days postinfection by qRT-PCR using TaqMan primer probe sets from Integrated DNA Technologies (IDT) (assay identifiers—Mm.PT.58.41769240 [IFNG], Mm.PT.58.10005566 [IL-6], Mm.PT.58.42155916 [GZMB], Mm.PT.58.41616450 [IL-1β], Mm.PT.58.43575827 [CXCL10], and Mm.PT.58.13531087 [IL-10]). Relative expression for each cytokine was determined by 2−ΔΔCT analysis with fold induction being relative to cytokine levels in brain RNA extracts of naive Ifnar1−/− mice.
3
Quantitative RT-PCR for Gene Expression
4
Quantitative RT-PCR for mRNA Expression
5
Cytokine Expression in Obesity Models
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Blood from naïve wild type and obese female and male mice that had been fed their respective diets for 12 weeks was collected into RNAzol BD (Molecular Research Center, Inc.: RB 192) and RNA was isolated according to the manufacturer’s instructions. mRNA expression of TNF-α, IL-1β and IL-6 was determined through qRT-PCR using Taqman primer probe sets purchased from Integrated DNA Technologies (IDT) based on the following assay identifiers: Mm.PT.58.12575861 (TNF-α), Mm.PT.58.41616450 (IL-1β) and Mm.PT.58.10005566 (IL-6). Relative expression of each cytokine was determined by 2ΔΔCT with fold induction being relative to GAPDH (assay identifier: Mm.PT.39a.1) levels of the same samples.
6
Quantitative Analysis of Gene Expression
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Liver, skeletal muscle, and uterine tissues were homogenized in 1mL of TRIzol reagent and then total RNA was isolated. Total RNA was reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster, CA, USA). cDNA was used as a template for the relative quantitation of selected target genes with predesigned TaqMan primer/probe sets (Integrated DNA Technologies, Coralville, IA, USA). Each 20 μL reaction mixture contained 100 ng cDNA, 2× SensiFAST Probe Lo-ROX mix (Bioline, Taunton, MA, USA), and TaqMan primer/probe. All reactions were carried out in triplicate with the 7500 Real-Time PCR Systems (Applied Biosystems) under the following conditions: 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Results were expressed as a relative value after normalization to β-actin.
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