Taqman fast universal pcr master mix 2x no amperase ung

Manufactured by Thermo Fisher Scientific

Sourced in United States

TaqMan™ Fast Universal PCR Master Mix (2X) no AMPERASE™ UNG is a pre-formulated, ready-to-use solution for real-time PCR. It contains all the necessary components, except primers and probes, to perform fast, sensitive, and reproducible real-time PCR reactions.

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TaqMan Fast Universal Master Mix (2X) No AmpErase UNG Taqman 2X universal PCR master mix (no AmpErase UNG) 2X TaqMan Universal PCR master mix no AmpErase UNG TaqMan® Universal PCR Master Mix No AmpErase TaqMan Fast Universal PCR Master Mix (2X) TaqMan Fast Universal Master Mix 2X TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2X TaqMan Universal Master Mix 2X TaqMan PCR Master Mix 2X

7 protocols using taqman fast universal pcr master mix 2x no amperase ung

Rapid Real-Time PCR Amplification

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Real-time PCR was run in fast mode by a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), using the following cycling conditions and thermal profile: 95 °C for 20 s, 40 cycles with 95 °C for 3 s, and 60 °C for 30 s. The reaction volume was 20 µL, with 1X of TaqMan^® Fast Universal PCR Master Mix (2X) No AmpErase UNG (Thermo Fisher Scientific, Waltham, MA, USA), 2 µL of DNA, and primers/probes at concentrations obtained in optimisation test. Data analysis was performed by SDS 2.4 software (Applied Biosystems^®, Foster City, CA USA). Each PCR run included not template control (NTC) and positive amplification control.

Torricelli M., Pierboni E., Rondini C., Altissimi S, & Haouet N. (2020). Sesame, Pistachio, and Macadamia Nut: Development and Validation of New Allergenic Systems for Fast Real-Time PCR Application. Foods, 9(8), 1085.

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Quantifying Relative Gene Expression via RT-qPCR

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Relative genetic expression was determined using real‐time quantitative polymerase chain reaction (RT‐qPCR) (CFX96™ Real‐Time System, Bio‐Rad, CA, USA) using commercially available TaqMan primers (Tables 1, 2, 3) and TaqMan™ Fast Universal PCR Master Mix (2X) no AMPERASE™ UNG (ThermoFisher Scientific, Catalog# #4367846, MA, USA). Amplification was done in 40 cycles with the following conditions (Denaturing at 95°C for 20 seconds and annealing and extending at 60°C for 20 s). Cycle threshold values were normalized using Pan Eukaryotic 18S (ThermoFisher Scientific Cat# 4333760F, MA, USA), transformed using the 2^−∆CT method, and graphed to represent the relative genetic expression by sample, gene group, and species.

Rosado‐Franco J.J., Ellison A.L., White C.J., Price A.S., Moore C.F., Williams R.E., Fridman L.B., Weerts E.M, & Williams D.W. (2024). Roadmap for the expression of canonical and extended endocannabinoid system receptors and metabolic enzymes in peripheral organs of preclinical animal models. Physiological Reports, 12(4), e15947.

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Relative Gene Expression Analysis via RT-qPCR

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Relative genetic expression was determined using Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) (CFX96^™ Real-Time System, Bio-Rad, CA, USA) using commercially available TaqMan primers (Tables 1–3) and TaqMan^™ Fast Universal PCR Master Mix (2X) no AMPERASE^™ UNG (ThermoFisher Scientific, Catalog#4367846, MA, USA). Amplification was done in 40 cycles with the following conditions (Denaturing at 95°C for 20 seconds and annealing and extending at 60°C for 20 seconds). Cycle threshold values were normalized using Pan Eukaryotic 18S (ThermoFisher Scientific, MA, USA), transformed using the 2^−∆CT method, and graphed to represent the relative genetic expression by sample, gene group and species.

Rosado-Franco J., Ellison A., White C., Price A., Moore C., Williams R., Fridman L., Weerts E, & Williams D. (2023). Roadmap For The Expression Of Canonical and Extended Endocannabinoid System Receptors and Proteins in Peripheral Organs of Preclinical Animal Models. bioRxiv.

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Quantitative Analysis of Reg3 Genes in SILP

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αβ T cells, γδ T cells, and epithelial cells were sorted from the SILP (ab and gd T cells) and epithelial layer (αβ, γδ T cells, and epithelial cells) and lysed in RLT buffer (Qiagen). Lysate was centrifuged for 3 min at maximum speed at 4 °C and transferred to a genomic DNA eliminator column. RNA extraction was performed as per the manufacturer’s instructions (RNeasy Plus Micro Kit, Qiagen). Next, RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific). Quantitative real-time PCR (RT-qPCR) was then performed on the cDNA using TaqMan Fast Universal PCR Master Mix (2X) no AmpErase UNG (Thermo Fisher Scientific) with a Vii 7 real-time PCR system (Applied Biosystems) with the following primers and probes: Reg3a (Mm01181787_m1), Re3b (Mm00440616_g1), and Reg3g (Mm00441127_m1). Quantitative PCR data were analyzed by the delta Ct method by normalizing the expression of each gene to Gapdh (Mm99999915_g1) or b-actin (Mm02619580_g1).

Rezende R.M., Cox L.M., Moreira T.G., Liu S., Boulenouar S., Dhang F., LeServe D.S., Nakagaki B.N., Lopes J.R., Tatematsu B.K., Lemos L., Mayrink J., Lobo E.L., Guo L., Oliveira M.G., Kuhn C, & Weiner H.L. (2023). Gamma-delta T cells modulate the microbiota and fecal micro-RNAs to maintain mucosal tolerance. Microbiome, 11, 32.

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Quantitative RT-PCR Analysis of Trem2 Expression

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Either frozen hippocampi from P1, P20, P90 or CA1/CA3 1-mm punches from Trem2^+/+ P18 mice were gently thawed on ice and then subjected to mechanical disruption with homogenizing beads onto automatic Tissue Lyser, in 500 μl TRI Reagent® (Zymo Research). RNA was isolated with RNA Direct-Zol™ MiniPrep Isolation Kit (Zymo Research), according to the manufacturer guidelines. RNA was eventually eluted in 25 μl DNAse/RNAse-free water and quantified with NANOdrop 2000c spectrophotometer (Thermo Fisher Scientific) for RNA concentration and 260/280 nm optical density ratios.
500 ng RNA for each condition underwent reverse transcription into cDNA with High-Capacity cDNA RT kit (Applied Biosystems). Quantitative Real-time polymerase chain reaction (qRT-PCR) was performed with TaqMan detection kit (TaqMan Fast Universal PCR Master Mix(2x), no AmpErase UNG, ThermoFisher) onto qRT-PCR Viia7 software system (Applied Biosystems) in a final volume of 10 μl. Each gene was subjected to at least duplicate measurements and data analyses were performed with the comparative ΔC_T method. mRNA measurements for each target gene were normalized to the housekeeping gene Gapdh. The following TaqMan assays were used (Applied Biosystems): mouse Trem2 FAM-MGB Mm04209424_g1; mouse GAPD(GAPDH) VIC-MGB Endogenous Control.

Tagliatti E., Desiato G., Mancinelli S., Bizzotto M., Gagliani M.C., Faggiani E., Hernández-Soto R., Cugurra A., Poliseno P., Miotto M., Argüello R.J., Filipello F., Cortese K., Morini R., Lodato S, & Matteoli M. (2024). Trem2 expression in microglia is required to maintain normal neuronal bioenergetics during development. Immunity, 57(1), 86-105.e9.

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Quantitative RT-PCR for Immune Genes

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RNA was extracted with the RNeasy Plus Mini Kit (#74136, Qiagen) or Quick-RNA MicroPrep Kit (#R1051, Zymo). Any remaining genomic DNA was removed by extraction on a column or by DNase digestion. RNA was reverse-transcribed with the SuperScript II Reverse Transcriptase (#18064014, Thermo Fisher Scientific) and oligo(dT)_12–18 (#18418012, Thermo Fisher Scientific) or with the High-Capacity RNA-to-cDNA Kit (#4387406, Applied Biosystems), according to the manufacturer’s protocol. qPCR was performed on cDNA with TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG (#4352042, Thermo Fisher Scientific) on a 7500 Real-Time PCR System (Applied Biosystems) or Taqman ViiA7, with the following probes, all from Thermo Fisher Scientific: IRF1 exons 3–4 (#Hs00971960_m1), IRF1 exons 8–9 (#Hs00971965_m1), GBP4 (#Hs00364728_m1), APOL3 (#Hs00758274_m1), and GUSB (#1702016).

Rosain J., Neehus A.L., Manry J., Yang R., Le Pen J., Daher W., Liu Z., Chan Y.H., Tahuil N., Türel Ö., Bourgey M., Ogishi M., Doisne J.M., Izquierdo H.M., Shirasaki T., Le Voyer T., Guérin A., Bastard P., Moncada-Velez M., Han J.E., Khan T., Rapaport F., Hong S.H., Cheung A., Haake K., Mindt B.C., Perez L., Philippot Q., Lee D., Zhang P., Rinchai D., Al Ali F., Ata M.M., Rahman M., Peel J.N., Heissel S., Molina H., Kendir-Demirkol Y., Bailey R., Zhao S., Bohlen J., Mancini M., Seeleuthner Y., Roelens M., Lorenzo L., Soudée C., Paz M.E., Gonzalez M.L., Jeljeli M., Soulier J., Romana S., L’Honneur A.S., Materna M., Martínez-Barricarte R., Pochon M., Oleaga-Quintas C., Michev A., Migaud M., Lévy R., Alyanakian M.A., Rozenberg F., Croft C.A., Vogt G., Emile J.F., Kremer L., Ma C.S., Fritz J.H., Lemon S.M., Spaan A.N., Manel N., Abel L., MacDonald M.R., Boisson-Dupuis S., Marr N., Tangye S.G., Di Santo J.P., Zhang Q., Zhang S.Y., Rice C.M., Béziat V., Lachmann N., Langlais D., Casanova J.L., Gros P, & Bustamante J. (2023). Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria. Cell, 186(3), 621-645.e33.

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Validation of Microarray Candidates by qRT-PCR

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Quantitative real-time PCR (qRT-PCR) was performed on a LightCycler^® 480 Instrument (Roche Applied Sciences, Penzberg, Germany). Validation of human microarray gene candidates from the same SPIA cDNA was performed using the TaqMan expression gene assay (Thermo Fischer Scientific) for the following genes: CNN1 (Cat.#Hs00959434_m1), SNRPN (Cat.#Hs01374551_m1), PLA2G7 (Cat.#Hs00965837_m1), ADAM20 (Cat.#Hs01083178_s1), MIR1183 (Cat.#Hs04273420_s1), and GAPDH (Cat.#Hs03929097_g1) along with TaqMan^™ Fast Universal PCR Master Mix (2X), no AmpErase^™ UNG (Thermo Fisher Scientific, Waltham, MA, USA). Verification of MIR1183 gene expression in AFIB and DCM tissue was conducted after cDNA synthesis using the QuantiTect Reverse transcription kit (Qiagen, Hilden, Germany) with the TaqMan^® Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). GenEx 5.4.4. Software (MultiD, Gothenburg, Sweden) had been used to correct primer efficiency followed by 2^−ΔΔct relative quantification. The results are presented as fold change in gene expression normalized to control.

Djalinac N., Kolesnik E., Maechler H., Scheruebel-Posch S., Pelzmann B., Rainer P.P., Foessl I., Wallner M., Scherr D., Heinemann A., Sedej S., Ljubojevic-Holzer S., von Lewinski D, & Bisping E. (2022). miR-1183 Is a Key Marker of Remodeling upon Stretch and Tachycardia in Human Myocardium. International Journal of Molecular Sciences, 23(13), 6962.

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