Taqman fast universal pcr master mix 2x

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

TaqMan Fast Universal PCR Master Mix (2X) is a pre-mixed solution that contains all the necessary components for performing real-time PCR reactions, including DNA polymerase, dNTPs, and buffer. It is designed to enable fast and efficient amplification of DNA targets.

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63 protocols using taqman fast universal pcr master mix 2x

Cardiac Gene and miRNA Expression

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Cardiac expression of Agtr2 (AT2R), Agtr1a (AT1A), Agtr1b (AT1B), miR-7a-1-3p, miR-138-5p, miR-148b-3p, and miR-101b-3p miRNAs were determined using mRNA and miRNA isolated from frozen ZO rat heart tissues (n = 5 per group) as described previously (Luck et al., 2017 (link); Lum-Naihe et al., 2017 (link); Belenchia et al., 2018 (link)). Agtr2, At1ra, At1rb, and 18s PCR reactions were performed in triplicate using TaqMan Fast Universal PCR Master Mix (2X) and TaqMan Assays with primers specific to the genes of interest (Applied Biosystems). miRNA real-time PCR reactions were performed in triplicate using either TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems) or miScript II Mix (Qiagen). TaqMan Advanced MicroRNA Assays (Life Technologies) primers were used for miR-7a-1-3p and miR-138-5p. miScript II Assay Primers (Qiagen) were used for miR-148-3p, miR-101b-3p, and miR-190a-3p.

Gavini M.P., Mahmood A., Belenchia A.M., Beauparlant P., Kumar S.A., Ardhanari S., DeMarco V.G, & Pulakat L. (2021). Suppression of Inflammatory Cardiac Cytokine Network in Rats with Untreated Obesity and Pre-Diabetes by AT2 Receptor Agonist NP-6A4. Frontiers in Pharmacology, 12, 693167.

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Validation of miRNA Expression by RT-qPCR

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miRNAs significantly differentially expressed by NGS were validated by RT-qPCR on an independent validation cohort of 93 samples in one center (Semmelweis University, 2nd Department of Internal Medicine). Reverse transcription of RNA was performed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) and individual TaqMan miRNA assays (CN: 4427975, 4440886; Thermo Fisher Scientific). Selected miRNAs were hsa-miR-30e-5p (ID: 002223), hsa-miR-223-3p (ID: 002295), hsa-miR-30d-5p (ID: 000420), and hsa-miR-7-5p (ID: 005723_mat). As reference miRNA, cel-miR-39 (ID: 000200) was used. Quantitative real-time PCR was performed by the TaqMan Fast Universal PCR Master Mix (2x) (CN: 4352042; Thermo Fisher Scientific) on a Quantstudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific) in accordance with the manufacturer's protocol for TaqMan miRNA assays with minor modifications (the end volume of the reaction was 15 μl, program of thermal cycler was the following: after 20 s on 95°C, 40 cycles of 95°C for 3 s and 60°C for 30 s). Negative control reactions contained no cDNA templates. Samples were always run in triplicate. For data evaluation, the dCt method [delta Ct (cycle threshold) value equals target miRNA's Ct minus internal control miRNA's Ct] was used by Microsoft Excel 2016 (Microsoft, Redmond, WA, USA).

Decmann A., Nyírö G., Darvasi O., Turai P., Bancos I., Kaur R.J., Pezzani R., Iacobone M., Kraljevic I., Kastelan D., Parasiliti-Caprino M., Maccario M., Nirschl N., Heinrich D., Reincke M., Patócs A, & Igaz P. (2019). Circulating miRNA Expression Profiling in Primary Aldosteronism. Frontiers in Endocrinology, 10, 739.

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Quantifying Citrus Leprosis Virus C Loads

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CiLV-C loads were assessed by RT-qPCR using a TaqMan® probe complementary to the viral p29 ORF (G.D. Arena, P.L. Ramos-González, M.A. Machado, J. Freitas-Astúa, unpublished data). Reaction mixes were prepared as recommended by the TaqMan® Fast Universal PCR MasterMix 2X kit manufacturer (Thermo Scientific, Waltham, MA, USA) and the amplifications were carried out in a 7500 Fast Real-Time PCR System device (Thermo Scientific, Waltham, MA, USA). Each sample was analyzed in triplicate and three template-free controls were performed for each primer pair. Cq-values were compared with a standard curve to determine absolute quantities of CiLV-C p29 molecules. Absolute p29 quantities in infected A. thaliana and C. sinensis were normalized using the expression levels of the species-specific SAND genes as references. Average of the p29 quantities of each time point were statistically compared with one-way ANOVA and Tukey's HSD (honest significant difference) tests (α < 0.05).

Arena G.D., Ramos-González P.L., Nunes M.A., Ribeiro-Alves M., Camargo L.E., Kitajima E.W., Machado M.A, & Freitas-Astúa J. (2016). Citrus leprosis virus C Infection Results in Hypersensitive-Like Response, Suppression of the JA/ET Plant Defense Pathway and Promotion of the Colonization of Its Mite Vector. Frontiers in Plant Science, 7, 1757.

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Quantifying T-Cell Subset Gene Expression

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To measure gene expression in T_H1/17, T_H17, T_H1, and DN cells sorted from frozen PBMC with qPCR, total RNA was isolated and digested with DNase I with the RNAqeous micro total RNA isolation kit. cDNA was synthesized with the SuperScript VILO master mix and pre-amplified for 14 cycles with the TaqMan preAmp master mix following the manufacture’s instruction. qPCR analysis was run and analyzed with the ViiA 7 Real-Time PCR System (Life Technologies) using the TaqMan fast universal PCR master mix (2x) and qPCR primers purchased from ThermoFisher Scientific. The comparative threshold cycle method and an internal control (β2m) were used for normalization of the target genes. Relative expression was calculated as: ΔC_T = C_{Tgene of interest} – C_Tβ2m; ΔΔC_T = ΔC_{T cell subset of interest} – mean of ΔC_{T DN of healthy control}; the relative change of gene expression between the expression level of sample of interest and the mean expression level of all DN samples in healthy controls was given by this formula: (2 ^–ΔΔCT) × 10. All qPCR reactions were performed in duplicate.

Hu D., Notarbartolo S., Croonenborghs T., Patel B., Cialic R., Yang T.H., Aschenbrenner D., Andersson K.M., Gattorno M., Pham M., Kivisakk P., Pierre I.V., Lee Y., Kiani K., Bokarewa M., Tjon E., Pochet N., Sallusto F., Kuchroo V.K, & Weiner H.L. (2017). Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis. Nature Communications, 8, 1600.

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GFP Expression Quantification in T Cells and Liver

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To detect GFP cDNA in T cells and liver samples we used TaqMan® Gene Expression Assays with two probe and primer sets, one for enhanced GFP (Mr04329676_mr Enhance) and mouse GAPDH (Mm99999915_g1) (ThermoFisher). We used 1 ul of the cDNA reaction in a qPCR mastermix using TaqMan™ Fast Universal PCR Master Mix (2x), no AmperErase™ UNG (ThermoFisher). Each cDNA sample was run separately in both a GAPDH and GFP Taqman probe and primer reaction in an ABI7500 Fast Thermalcycler.

Breuer C.B., Hanlon K.S., Natasan J.S., Volak A., Meliani A., Mingozzi F., Kleinstiver B.P., Moon J.J, & Maguire C.A. (2020). In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery. Scientific Reports, 10, 4544.

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Detailed Protocol for CD4+ T-cell Isolation and Cytokine Analysis

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EasySep human CD4⁺ T-cell enrichment kit (catalog number 19052) for CD4⁺ T-cell isolation was purchased from StemCell Technologies. FITC-conjugated anti-human IFN-γ (clone, B27; 1:100), Alexa 647-conjugated anti-human IL-17A (clone, N49-653; 1:20), PE-conjugated anti-human IL-10 (clone, JES3-19F; 1:660), and their corresponding isotype control antibodies for intracellular cytokine staining assay were purchased from BD Biosciences. IFN-γ cytokine secretion detection kit (APC) (catalog number 130-090-762) and IL-17 cytokine secretion detection kit (PE) (catalog number 130-094-537) were purchased from Miltenyi Biotec. nCounter CodeSet HuT_H17 was custom designed and manufactured by nanoString Technologies. Fluorescence-conjugated antibodies for cell surface staining for flow cytometry were purchased from Biolegend. RNAqeous micro total RNA isolation kit (catalog number AM1931), SuperScript VILO master mix (catalog number 11755050), TaqMan preAmp master mix (catalog number 4391128), TaqMan fast universal PCR master mix (2x) (catalog number 4352042), and qPCR primers (Supplementary Data 8) were purchased from ThermoFisher Scientific.

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Validating RNA-seq with qRT-PCR

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To verify some of the results obtained from the RNA sequencing, qRT-PCR was carried out as previously described [5 (link)]. Briefly, miRNA cDNA was prepared from each sample with 30 ng of template RNA using TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). For plasma samples, the same volume of spiked-in plasma RNA was directly used as a template. Gene expression was measured using standard fast conditions in StepOne Plus Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA) in triplicate reactions consisting of 2.25 µL of 1:7-diluted cDNA, 0.25 µL of TaqMan MicroRNA Assay (listed in Table S2), 2.5 µL of TaqMan Fast Universal PCR Master Mix (2X), no AmpErase™ UNG (Thermo Fisher Scientific). The CNS tissue samples were normalized with the mean of miR-369-5p and miR-186-5p and the plasma samples with the mean of miR-16-5p and spiked-in cel-miR-39-3p as these combinations showed the greatest stability among the samples when several candidates were tested by RefFinder (see also Appendix A, Figure A1) [20 (link)]. Relative expression between the prion-inoculated and the control mice was calculated as previously described [5 (link)].

Toivonen J.M., Sanz-Rubio D., López-Pérez Ó., Marín-Moreno A., Bolea R., Osta R., Badiola J.J., Zaragoza P., Espinosa J.C., Torres J.M, & Martín-Burriel I. (2020). MicroRNA Alterations in a Tg501 Mouse Model of Prion Disease. Biomolecules, 10(6), 908.

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Quantification of Cholinergic Markers in Spinal Cords

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Pups were euthanized with an overdose of Dolethal (20 mg/ml) on PND11. Lumbar spinal cords were dissected and homogenized in TRIzol (Thermo Fisher Scientific, 15596026) using the MagNaLyser oscillator. Total RNA was precipitated with isopropanol, of which 1 μg was used to prepare cDNA with the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, 18080051). Quantitative PCRs were performed with the TaqMAN Fast Universal PCR Master Mix 2X (Thermo Fisher Scientific, 4364103) using 1/10 diluted cDNA and the following Taqman assays (IDT): Chat (Mm01221882_m1), Gapdh (Mm.PT.39a.1) and Polr2a (Mm.PT.58.13811327). PCR reaction was performed in a StepOnePlus instrument (Life Technologies) and relative gene expression was analyzed with the Qbase + software (Biogazelle).

Poppe L., Smolders S., Rué L., Timmers M., Lenaerts A., Storm A., Schoonaert L., de Boer A., Van Damme P., Van Den Bosch L., Robberecht W, & Lemmens R. (2019). Lowering EphA4 Does Not Ameliorate Disease in a Mouse Model for Severe Spinal Muscular Atrophy. Frontiers in Neuroscience, 13, 1233.

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Validating miRNA Expression via qPCR

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Taqman assays (ThermoFisher Scientific) were used to validate the results obtained from MicroRNA micro-array analysis. Reverse transcription was carried out using reverse transcription kit (ThermoFisher Scientific #4366596) as per the manufacturer's protocol. Total RNA used per reaction for the reverse transcription step was 100ng. Determination of miRNA expression of miR-125a-5p and miR-145b was done by using TaqMan micro RNA assay (ThermoFisher Scientific Catalog # 4427975), and TaqMan Fast Universal PCR master mix 2x (ThermoFisher Scientific Catalog # 4352042) as per the manufacturer's instructions. The detection system used was CFX96 qPCR (BioRad). The reactions for the gene of interest were carried out in triplicates, while normalizing control was run in duplicates. Validation was done using the same RNA samples, which were used for micro array analysis. The comparative delta-Ct method was used to quantify the results. The normalizing control used was snoRNA202 (ThermoFisher Scientific #4427975).

Makwana K., Patel S.A., Velingkaar N., Ebron J.S., Shukla G.C, & Kondratov R.V. (2017). Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13. Aging (Albany NY), 9(7), 1825-1837.

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Profiling miRNA in EV and ASC-EV

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Total miRNA was collected from EVs and/or ASC EVs using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was prepared using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The expression levels of hsa-mir-218-5p (assay ID: 000521), hsa-mir-214 (assay ID: 002293), hsa-miR-195 (assay ID: 000494), and internal control U6snRNA (assay ID: 001973) (Table 2) were analyzed using the Thermal Cycler Dice^® Real-Time System (TP 800; Takara Bio, Shiga, Japan) and the TaqMan Fast Universal PCR Master Mix (2X) (4366072, Thermo Fisher Scientific, Waltham, MA, USA).

Kazi T., Nagata A., Nakagawa T., Matsuzaki T, & Inui S. (2022). Dermal Papilla Cell-Derived Extracellular Vesicles Increase Hair Inductive Gene Expression in Adipose Stem Cells via β-Catenin Activation. Cells, 11(2), 202.

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