Fam mgb probe

Manufactured by Thermo Fisher Scientific

Sourced in United States

FAM/MGB probes are fluorescent oligonucleotide probes used in real-time PCR assays. They are designed to detect and quantify specific DNA sequences. FAM/MGB probes utilize a fluorescent reporter dye (FAM) and a non-fluorescent quencher (MGB) to provide sensitive and specific detection of target sequences.

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TaqMan FAM/MGB probes FAM-NFQ MGB probes FAM-labeled MGB Taqman probes FAM-MGB labeled Taq-Man probes FAM dye-labeled MGB-probes FAM-MGB-conjugated TaqMan primer probes TaqMan Gene Expression Assays (FAM dye-labeled MGB probe; Human GAPDH Endogenous Control FAM™ Dye/MGB Probe FAM dye-labeled TaqMan MGB probes TaqMan® gene expression FAM/MGB probe system

14 protocols using fam mgb probe

Quantitative analysis of gene expression

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Total RNA was isolated using a Trizol. cDNA was generated by using 100 ng of total RNA, and TaqMan quantitative PCR analysis of Il23r, Cox20, and Gapdh mRNA expression was conducted using commercially available primers and FAM/MGB probes (Applied Biosystems). Data were reported as relative expression normalized to the housekeeping gene Gapdh. In select experiments focused on Let-7f expression levels, miRNA was amplified per manufacturer's directions using the Quantabio qScript miRNA 2-step qPCR kit and commercially available primers and FAM/MGB probes (Applied Biosystems). Data were reported as relative expression normalized to the housekeeping gene U6B.

Fuseini H., Cephus J.Y., Wu P., Davis J.B., Contreras D.C., Gandhi V.D., Rathmell J.C, & Newcomb D.C. (2019). ERα Signaling Increased IL-17A Production in Th17 Cells by Upregulating IL-23R Expression, Mitochondrial Respiration, and Proliferation. Frontiers in Immunology, 10, 2740.

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Pathogen Copy Number Estimation in CSF

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Copy number of host and spiked pathogen NA in native surrogCSF and its change with host NA depletion were estimated using qPCR. The primer systems used were ACTB human (Hs01060665_g1) and Eukaryotic 18S rRNA Endogenous Control (FAM™/MGB probe, non-primer limited) (Thermo Fisher Scientific Inc.), the mitochondrial NADH dehydrogenase subunit 1 (ND1) primer-probe set by He et al. (2002 (link)), the validated in-house primer-probe set InfAM (M-protein) for Inf A, and the validated in-house primer-probe set entF3 for Y. pseud. The qPCR analyses were performed on a LightCycler® 96 System (Roche Diagnostics International AG) using the TaqMan™ Fast Advanced Master Mix for DNA and TaqMan® Fast Virus 1-Step Master Mix for RNA analysis (Applied Biosystems™, Thermo Fisher Scientific Inc.). The run protocols of the respective master mixes for 45 cycles were: Fast Virus 1-Step: 300 s at 50°C, 20 s at 95°C, 45x (3 s at 95°C, 30 s at 60°C), Fast Advanced: 20 s at 95°C, 45x (3 s at 95°C, 30 s at 60°C). Negative qPCR results in host NA depletion evaluations were set to a Ct-value 42 for calculation purposes. Copy numbers are estimated given a Ct-value of 21 corresponding to a copy number of 10E+5 (slope of −3.33/log).

Oechslin C.P., Lenz N., Liechti N., Ryter S., Agyeman P., Bruggmann R., Leib S.L, & Beuret C.M. (2018). Limited Correlation of Shotgun Metagenomics Following Host Depletion and Routine Diagnostics for Viruses and Bacteria in Low Concentrated Surrogate and Clinical Samples. Frontiers in Cellular and Infection Microbiology, 8, 375.

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Quantitative Analysis of Gene Expression

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Cells were prepared for RNA extraction with the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Reverse transcription was performed using SUPERSCRIPT II (18064071, Thermo Fisher Scientific). Gene expression was measured using the following TaqMan Gene Expression Assays along with TaqMan Gene Expression Master Mix (4369016, Thermo Fisher Scientific): human B2M (Beta-2-Microglobulin) Endogenous Control (VIC/MGB probe, primer limited) (4326319E, Thermo Fisher Scientific), human PAH Exon Boundary 1-2 (Hs07288474_m1 FAM/MGB probe, Thermo Fisher Scientific), human PAH Exon Boundary 7-8 (Hs07288479_m1, FAM/MGB probe, Thermo Fisher Scientific). Each 10 μL qRT-PCR reaction contained 5 μL TaqMan Gene Expression Master Mix, 0.5 μL B2M probe, 0.5 μL probe to target the gene of interest, and 4 μL cDNA (diluted 1:10 with water) and was performed in technical duplicates. Reactions were carried out on the QuantStudio 7 Flex System (Thermo Fisher Scientific). Relative expression levels were quantified by the 2⁻^ΔΔCt method.

Brooks D.L., Carrasco M.J., Qu P., Peranteau W.H., Ahrens-Nicklas R.C., Musunuru K., Alameh M.G, & Wang X. (2023). Rapid and definitive treatment of phenylketonuria in variant-humanized mice with corrective editing. Nature Communications, 14, 3451.

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Quantitative Analysis of Lung Microbiome

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Following the BALF procedure (see below), the left lobe of each lung was minced using a sterile scalpel blade. A 2 mm³ sample of the lung was snap-frozen in liquid nitrogen. DNA was extracted from lung samples using the DNeasy Blood and Tissue DNA extraction kit as described by the manufacturer (Qiagen). Universal 16S rRNA primers (F primer = 5′-TCCTACGGGAGGCAGCAG T-3′; R primer = 5′-GGACTACCAGGGTATCTAATCCTGTT-3′ [26 (link)]) were used to detect bacteria in DNA samples using the Power SYBR Green PCR Master Mix as described by the manufacturer (Applied Biosystems, Calsbad, USA). These ‘universal primers’ have broad specificity to detect conserved regions of 16S rDNA from 34 bacterial species encompassing most bacterial groups [26 (link)]. An 18S rRNA endogenous control including a FAM-MGB probe was used as the internal control for this assay using conditions described by the manufacturer (Applied Biosystems).

Roggenbuck M., Anderson D., Barfod K.K., Feelisch M., Geldenhuys S., Sørensen S.J., Weeden C.E., Hart P.H, & Gorman S. (2016). Vitamin D and allergic airway disease shape the murine lung microbiome in a sex-specific manner. Respiratory Research, 17, 116.

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Quantifying CYP1A1 and CYP1B1 Induction

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Mammary tissues (100 mg) were homogenized in TRIzol reagent. The total RNA was extracted using the RNeasy lipid tissue kit
(Qiagen, Valencia, CA) and RNA (5 μg) was reverse transcribed with Invitrogen’s SuperScript® III First-Strand
Synthesis System. RT-qPCR was performed using TaqMan rat CYP1A1, CYP1B1, and ACTB primers with FAM/MGB probe (Applied Biosystems,
Carlsbad, CA) as described previously (29 (link)). MCF-10A cells were obtained from ATCC
(Manassas, VA) and authenticated via STR profiling (Promega). MCF-10A cells were cultured as described previously (29 (link)). For the in vitro CYP1A1/CYP1B1 induction experiments, cells having approximately
15 through 20 passages were plated in 96-well plates and treated with vehicle (DMSO), GG, GI, and licorice compounds for 24 h.
RT-qPCR was performed as previously described (29 (link)) using TaqMan® 1-Step RT-PCR
Master Mix, and CYP1A1 and CYP1B1 primer with FAM-MGB probe and GAPDH primer with VIC-MGB probe. Data were analyzed with the
comparative C_T (ΔΔC_T) method and expressed as fold induction
relative to the vehicle control group.

Wang S., Dunlap T.L., Huang L., Liu Y., Simmler C., Lantvit D.D., Crosby J., Howell C.E., Dong H., Chen S.N., Pauli G.F., van Breemen R.B., Dietz B.M, & Bolton J.L. (2018). Evidence for chemopreventive and resilience activity of licorice: Glycyrrhiza glabra and G. inflata extracts modulate estrogen metabolism in ACI rats. Cancer prevention research (Philadelphia, Pa.), 11(12), 819-830.

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Quantification of Bovine Immune Gene Expression

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TaqMan bovine gene expression assays (Applied Biosystems, Foster City, CA) listed in Table 1 were used to quantify relative expression of IL-1β (IL1B), IL-10 (IL10), IL-12A (IL12A), IL-17A (IL17A), β-defensin 7 (DEFB7), β-defensin 10 (DEFB10), CYP24A1 (CYP24A1), CYP27B1 (CYP27B1), IFN-γ (IFNG), iNOS (NOS2), RANTES (CCL5), and TNF-α (TNF) in cells from the 24 hr cytokine supernatant samples. The following protocol was optimized previously in our lab¹. Samples were plated in duplicate with a reaction mixture consisting of 10 μl TaqMan Fast Advanced Master Mix (Applied Biosystems), 1 μl gene expression assay, 5 μl nuclease-free water, and 4 μl cDNA template per well. Relative quantitation (RQ) values were calculated by normalization to 18S rRNA expression (FAM/MGB probe, non-primer limited; Applied Biosystems) and calibration to the NS sample. Data were analyzed using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).

Wherry T.L., Dassanayake R.P., Casas E., Mooyottu S., Bannantine J.P, & Stabel J.R. (2022). Exogenous Vitamin D3 Modulates Response of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis Infection and Is Dependent Upon Stage of Johne’s Disease. Frontiers in Cellular and Infection Microbiology, 11, 773938.

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Confirming Cd247 mRNA Expression by qRT-PCR

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The differential expression of Cd247 mRNA as observed by microarray hybridizations was confirmed using qRT-qPCR. Complementary DNA (cDNA) was synthesized from 500 ng of total RNA using High-Capacity RNA-to-cDNA Kit (Applied Biosystems—Life Technologies, USA) as recommended. The PCR products were amplified from cDNA samples using Applied Biosystems TaqMan Universal PCR Master Mix (Applied Biosystems—Life Technologies, USA) with an assay probe spanning the exon junction of the murine Cd247 (FAM/MGB probe; assay ID: Mn00446171_m1 from Applied Biosystems—Life Technology, USA) or for the Applied Biosystems mouse GAPDH endogenous control (FAM/MGB probe, Non-Primer Limited; assay ID: Mm00446171_m1) that was used as constitutive reference.

Fornari T.A., Donate P.B., Assis A.F., Macedo C., Sakamoto-Hojo E.T., Donadi E.A, & Passos G.A. (2015). Comprehensive Survey of miRNA-mRNA Interactions Reveals That Ccr7 and Cd247 (CD3 zeta) are Posttranscriptionally Controlled in Pancreas Infiltrating T Lymphocytes of Non-Obese Diabetic (NOD) Mice. PLoS ONE, 10(11), e0142688.

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Quantifying miRNA and mRNA Expression

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Real-time PCR assays to measure mRNA expression levels of miR-200c, E-cadherin, and CD44 were performed as published elsewhere.23 (link) Total RNA was harvested from cells using TRIzol^® reagent (Life Technologies, Carlsbad, CA, USA). The isolated RNA was reverse transcribed into complementary DNA using an ExScript™ RT reagent kit (Takara Bio Inc, Otsu, Japan) according to the manufacturer’s instructions. The reaction was allowed to proceed at 25°C for 10 minutes and then at 37°C for one hour. For normalization, reverse transcription PCR was done on complementary DNA using eukaryotic 18S rRNA endogenous control primers and a FAM-MGB probe (Applied Biosystems, Foster City, CA, USA). A TaqMan miRNA reverse transcription kit was used to generate complementary DNA for PCR reaction in conjunction with a miR-200c-specific primer and probe (Applied Biosystems, assay ID 002300). The reverse transcription primer for miR-200c is a hairpin primer that is specific for mature miRNA and does not bind to the precursor molecules. For validation of the microarray data, SYBR Green real-time reverse transcription PCR was done using primers specific for E-cadherin and CD44. The specific primer sequences are shown in Figure S3. Relative mRNA and miRNA levels were calculated using the comparative Ct method (ΔΔCt).

Cui F.B., Liu Q., Li R.T., Shen J., Wu P.Y., Yu L.X., Hu W.J., Wu F.L., Jiang C.P., Yue G.F., Qian X.P., Jiang X.Q, & Liu B.R. (2014). Enhancement of radiotherapy efficacy by miR-200c-loaded gelatinase-stimuli PEG-Pep-PCL nanoparticles in gastric cancer cells. International Journal of Nanomedicine, 9, 2345-2358.

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RT-qPCR Analysis of Gene Expression

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The RT-qPCR analysis was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems, Stockholm, Sweden). Total RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen, Sollentuna, Sweden), cDNA was synthesized using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA), and FAM/MGB probes (Applied Biosystems, Foster City, CA, USA) were used for the PCR reaction. Amplification of two housekeeping genes, GAPDH and ß-actin was used as an internal standard. The comparative C_t method was applied to determine the fold-difference in expression levels relative to a control sample.

Wiechec E., Hansson K.T., Alexandersson L., Jönsson J.I, & Roberg K. (2017). Hypoxia Mediates Differential Response to Anti-EGFR Therapy in HNSCC Cells. International Journal of Molecular Sciences, 18(5), 943.

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Quantifying mRNA Downregulation by siRNA

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Total RNA was extracted with the RNeasy Mini Kit (Qiagen), and cDNA was obtained with the High Capacity RNA-to-cDNA Kit (Applied Biosystems). The expression levels of TSG101 or VPS4 mRNA were analysed with a 7500 Fast Real-Time PCR system and FAM/MGB probes (Applied Biosystems) to confirm downregulation after siRNA treatment. All reactions were performed according to the manufacturer’s instructions. GAPDH was amplified as an internal standard. The data were calculated according to the comparative Ct method to present the data as fold differences in the expression levels relative to the control sample.

Sardar Sinha M., Ansell-Schultz A., Civitelli L., Hildesjö C., Larsson M., Lannfelt L., Ingelsson M, & Hallbeck M. (2018). Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathologica, 136(1), 41-56.

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