Taqman pcr master mix

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Belgium

TaqMan PCR Master Mix is a ready-to-use solution for real-time PCR amplification. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform the PCR reaction.

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TaqMan Master Mix (448 citations) PCR Master Mix (310 citations) TaqMan® Real-Time PCR Master Mixes (7 citations)

249 protocols using taqman pcr master mix

Quantitative RT-PCR Analysis of Gene Expression

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mRNA was extracted from lung tissue using the Qiagen RNA extraction kit (74104, Qiagen) and reverse transcribed with SuperScript III Reverse Tran- scriptase (Life Technologies). TaqMan primers for gene expression assays were purchased from Life Technologies. Real-time qPCR was carried out with an ABI PRISM 7300 Sequence Detection System using TaqMan PCR Master Mix (Life Technologies). mRNA was extracted from cells using the Qiagen RNA extraction kit (74104, Qiagen) and reverse transcribed with SuperScript III Reverse Transcriptase (Life Technologies). mRNA in Supplementary Fig. 4f was used from RIP-Seq library preparations. TaqMan primers for human transferrin Receptor 1, human ferritin Heavy chain, human frataxin and human beta-actin for gene expression assays were purchased from Life Technologies. Real-time qPCR was carried out with an ABI PRISM 7300 Sequence Detection System using TaqMan PCR Master Mix (Life Technologies).

Cloonan S.M., Glass K., Laucho-Contreras M.E., Bhashyam A.R., Cervo M., Pabón M.A., Konrad C., Polverino F., Siempos I.I., Perez E., Mizumura K., Ghosh M.C., Parameswaran H., Williams N.C., Rooney K.T., Chen Z.H., Goldklang M.P., Yuan G.C., Moore S.C., Demeo D.L., Rouault T.A., D’Armiento J.M., Schon E.A., Manfredi G., Quackenbush J., Mahmood A., Silverman E.K., Owen C.A, & Choi A.M. (2016). Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice. Nature medicine, 22(2), 163-174.

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Quantitative RT-PCR Analysis of Gene Expression

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Quantitative PCR Analysis of Gene Expression

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Total RNA was extracted from cell-injected ischemic hindlimb tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. First-strand cDNA was generated using the Multiscribe Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Gene expression was determined by real-time quantitative PCR (7500 Fast Real-Time PCR System, Applied Biosystems) using TaqMan PCR Master Mix (Applied Biosystems). Relative mRNA expression of target gene normalized to that of glyceraldehye-3-phosphate dehydrogenase (GAPDH) was calculated using the formula: Relative Expression Level = 2^−ΔCT, where ΔCT = CT ^gene^{of interest} – CT ^GAPDH as previously described [7 (link)]. The primers and probes were designed using Primer Express 3.0 (Applied Biosystems) and are described in Table 1.

Lee S., Valmikinathan C.M., Byun J., Kim S., Lee G., Mokarram N., Pai S.B., Um E., Bellamkonda R.V, & Yoon Y. (2015). Enhanced Therapeutic Neovascularization by CD31-Expressing Cells and Embryonic Stem Cell-Derived Endothelial Cells Engineered with Chitosan Hydrogel Containing VEGF-Releasing Microtubes. Biomaterials, 63, 158-167.

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Evaluating Chondrogenic Gene Expression

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After treatment, sponges seeded with cells were rinsed once with ice-cold phosphate-buffered saline and total RNA was extracted using Trizol Reagent according to manufacturer’s instructions. One microgram of RNA was reverse transcribed into cDNA using reverse transcriptase (MMLV, Invitrogen) and oligodT (Eurogentec). PCR was performed on an Applied Biosystems 7700 Real-Time system using the TaqMan PCR Master Mix (Applied Biosystems) for COL2B collagen and using Power SYBR Green PCR (Applied Biosystems) for the other genes, as previously described. Sequences of the primers and probe used are listed in Table 2. Relative gene expression was calculated using the 2^−ΔΔCT method and expressed as the mean of triplicate samples. Each sample was normalized to RPL13a and each group was normalized to the expression levels of undifferentiated hUCB-MSCs.

Gómez-Leduc T., Hervieu M., Legendre F., Bouyoucef M., Gruchy N., Poulain L., de Vienne C., Herlicoviez M., Demoor M, & Galéra P. (2016). Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering. Scientific Reports, 6, 32786.

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Real-Time PCR Quantification of Gene Expression

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Total RNAs were isolated using TRIzol reagent (Invitrogen), from which cDNAs were synthesized using the MultiScribe Reverse Transcriptase (Applied Biosystems). Real-time PCR was performed using an ABI prism 7700 sequence detection systems (Applied Biosystems). Assays-on-Demand probes (Applied Biosystems) for PCR with the TaqMAN PCR Master Mix (Applied Biosystems) were: human CYP3A4 gene, Hs00604506_m1; human PEPCK1 gene, Hs00159918_m1; human G6Pase gene, Hs00609178_m1; mouse PEPCK1 gene, Mm00440636_m1. The TaqMan human and mouse glyceraldehyde-3-phosphate dehydrogenase (Applied Biosystems) was used as internal control.

Gotoh S, & Negishi M. (2015). Statin-activated nuclear receptor PXR promotes SGK2 dephosphorylation by scaffolding PP2C to induce hepatic gluconeogenesis. Scientific Reports, 5, 14076.

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RNA Isolation, cDNA Generation, and RT-PCR Analysis

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RNA was isolated from ileum using mechanical homogenization and TRIzol isolation (Invitrogen) according to the manufacturer’s instructions. cDNA was generated using the QuantiTect Reverse Transcription Kit (Qiagen). RT-PCR was performed on cDNA using TaqMan primers and probes in combination with TaqMan PCR Master Mix (Applied Biosystems), and reactions were run on an RT-PCR system (StepOne Plus; Applied Biosystems). Gene expression is displayed as fold increase over wild-type control mice, unless otherwise stated, and normalized to Hprt.
The extracted protein samples were run for the presence of Reg3γ and β-tubulin protein as described in (37 (link)). Briefly, identical amounts of protein were loaded on a 4 to 12% SDS-PAGE (polyacrylamide gel electrophoresis) gel and transferred to a nitrocellulose membrane, and rabbit polyclonal Reg3γ-specific antiserum or mouse anti–β-tubulin antibodies (Santa Cruz Biotechnology) were used to detect Reg3γ and the loading control protein. Original blot images are shown in Fig. S7. Bands were quantified using the ImageJ software, and the relative density values are listed in table S1.

Abt M.C., Buffie C.G., Sušac B., Becattini S., Carter R.A., Leiner I., Keith J.W., Artis D., Osborne L.C, & Pamer E.G. (2016). TLR-7 activation enhances IL-22–mediated colonization resistance against vancomycin-resistant enterococcus. Science translational medicine, 8(327), 327ra25.

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Quantitative RT-PCR analysis of cell cycle genes

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TaqMan primers for CDK4, CDK6, CCND1, CCNE1, CCNE2, CCNA2, E2F1, RB1, ESR1, PGR, GREB1, XBP1, TFF1 and MYC were used to detect mRNA levels by Quantitative RT-PCR. Total cellular RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cDNA was synthesized from 1 µg of RNA with the qScript cDNA SuperMix (Quanta Biosciences, Beverly, MA) and 2µl of cDNA was added to the Taqman PCR master Mix (Applied Biosystems, Foster City, CA) along with the appropriate primer. Relative quantification for each mRNA was performed using the comparative CT method with a ViiA 7 Real-Time PCR system (Applied Biosystems). The mRNA expression of the each gene was normalized to that of RPLP0. TaqMan primers were all purchased from Applied Biosystems. Each experiment was repeated at least three times with three replicates.

Yang C., Li Z., Bhatt T., Dickler M., Giri D., Scaltriti M., Baselga J., Rosen N, & Chandarlapaty S. (2016). Acquired CDK6 amplification promotes breast cancer resistance to CDK4/6 inhibitors and loss of ER signaling and dependence. Oncogene, 36(16), 2255-2264.

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Quantitative Analysis of Renal and Intestinal Ion Transporters

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Total RNA was extracted from the kidney and intestine (duodenum) with ISOGEN (Nippon Gene). cDNA was synthesized using the SuperScript III First Strand System (Invitrogen). Real-time quantitative reverse transcription-PCR (RT-PCR) was performed by incubating the reverse transcribed product with Taqman PCR Master Mix and a designed Taqman probe (ATP2B1: Mm01232254_m1, ATP2B4: Mm01285597_m1, Na⁺/Ca²⁺ exchanger 1 (NCX1): Mm01232254_m1, Transient Receptor Potential Vanilloid (TRPV) channel type6: Mm00499069_m1, renin: Mm02342887_mH, Applied Biosystems). RNA amounts are shown relative to the mRNA control (18s: 4319413E; Applied Biosystems).

Ehara Y., Hirawa N., Sumida K., Fujiwara A., Kagimoto M., Ooki-Okuyama Y., Fujita M., Katsumata M., Kobayashi Y., Saka S., Katou I., Yatsu K., Umemura S, & Tamura K. (2018). Reduced secretion of parathyroid hormone and hypocalcemia in systemic heterozygous ATP2B1-null hypertensive mice. Hypertension Research, 41(9), 699-707.

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Silencing TOX and PLS3 Expression

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EXAMPLE 3

5 ng of TOX Silencer Select Validated siRNA (Ambion, Carlsbad, Calif.) was added along with 1.5 μL of TransIT-siQUEST Transfection Reagent (Mirus, Madison, Wis.) per manufacturer's instruction to cultured PBMCs from patients 24 hours after culture was initiated. 72 hours later, RNA was isolated from treated cells using RNAprotect and RNeasy (Qiagen, Valencia, Calif.) per manufacturer's instructions. For cDNA synthesis, total RNA (100 ng) was used for Reverse Transcription (RT) with Superscript II reverse transcriptase (Life Technologies, Gaithersburg, Md.) using oligo dT primers according to the recommendations of the manufacturer. 2 μl of the resulting cDNA was used for each PCR reaction. Quantitative reverse transcription PCR was performed using TaqMan PCR master mix (Applied Biosystems, Foster City, Calif.) together with TaqMan probes and primers (Applied Biosystems) using standard conditions.

As shown in FIG. 6, siRNA knockdown of TOX resulted in decreased TOX expression and increased RUNX3 expression. siRNA knockdown of PLS3 also was found to decrease PLS3 expression.

US10473661B2. Method for diagnosis, prognosis and determination of treatment for cutaneous T-cell lymphoma (2019-11-12). UNIVERSITY OF PITTSBURGH—OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION [US]. Inventors: Larisa J. Geskin [US], Brittany Lynn O'Neil Dulmage [US], Louis D. Falo, Jr. [US].

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Quantitative Gene Expression Analysis

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Relative levels of mRNA from genes of cytokines and caspases were evaluated by real-time quantitative polymerase chain reaction (qPCR). Thymus RNA was extracted individually, converted to cDNA using commercial kits (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA) and RT-PCR reactions were carried out with TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA), according to manufacturer’s recommendations, using different sets of oligonucleotides and probes for amplification of the following mRNA: Glyceraldehyde 3-phosphate dehydrogenase GAPDH (endogenous control), interleukin (IL)-1β, IL-4, IL-6, IL-17, IL-18, IFN-γ, TNF-α, transforming growth factor-beta (TGF-β), caspase-1, caspase-3, caspase-8 and NLRP3 genes. These corresponded respectively to the following reference numbers (Applied Biosystems): Mm99999915_g1, Mm00434228_m1, Mm00445259_m1, Mm00446190_m1, Mm00439619_m1, Mm00434226_m1, Mm00801778_m1, Mm00443258_m1, Mm01178820_m1, Mm00438023_m1, Mm01195085_m1, Mm00802247_m1 and Mm00840904_m1. Data are presented as relative mRNA levels calculated by using the method of delta threshold (2^-ΔΔCt). ΔCt (ΔCt = Ct of target gene minus Ct of GAPDH).

Alves da Costa T., Di Gangi R., Thomé R., Barreto Felisbino M., Pires Bonfanti A., Lumi Watanabe Ishikawa L., Sartori A., Burger E, & Verinaud L. (2016). Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis. PLoS ONE, 11(10), e0164745.

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