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Taqman one step rt pcr kit

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The TaqMan One-Step RT-PCR kit is a reagent system for performing reverse transcription and real-time PCR amplification in a single reaction. The kit includes all the necessary components for the reverse transcription and amplification of RNA targets.

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Lab products found in correlation

Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Megascript, by Thermo Fisher Scientific (2 mentions) Maxiscript, by Thermo Fisher Scientific (2 mentions) Icycler software, by Bio-Rad (1 mentions) Icycler iq, by Bio-Rad (1 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions) Rneasy kit, by Qiagen (1 mentions) Specific primers and probes, by Thermo Fisher Scientific (1 mentions) Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions) Steponeplus detection system, by Thermo Fisher Scientific (1 mentions) Tri reagent, by Molecular Research Center (1 mentions) Col1a1, by Thermo Fisher Scientific (1 mentions) Steponeplus system, by Thermo Fisher Scientific (1 mentions) Qpcr primers and probes, by Thermo Fisher Scientific (1 mentions) Fgf23, by Thermo Fisher Scientific (1 mentions) Cyp27b1, by Thermo Fisher Scientific (1 mentions) Cyp24a1, by Thermo Fisher Scientific (1 mentions) Qiaamp viral rna mini kit, by Qiagen (1 mentions) Taqman primer, by Thermo Fisher Scientific (1 mentions) 7500 real time pcr stepone plus system and software, by Thermo Fisher Scientific (1 mentions)

19 protocols using taqman one step rt pcr kit

1

Quantitative RT-PCR Assay for In Vitro Splicing

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Based on the protocol outlined in [36 (link)], aliquots of in vitro splicing reactions were immediately diluted 1:65 in water, vortexed and kept on ice until all samples were ready for analysis. Using the TaqMan® One-Step RT-PCR kit (Applied Biosystems), 2 μl of the diluted splicing reactions were added in triplicate samples as template to 10 μl of reactions that included primers to ends of the exons of the pre-mRNA substrate and TaqMan probe to the exon junction of spliced mRNA. Experiments were performed and analysed using an iCycler iQ (BioRad). iCycler software (BioRad) was used to generate threshold cycles (Ct) for each well; triplicate samples were averaged and samples with S.D. more than one cycle were tagged for manual inspection. If an outlier was found, the average of duplicate samples was used for downstream analysis. To determine ΔCt, triplicate-averaged Ct for each protein-added sample was compared with triplicate-averaged buffer-only splicing from the same experiment. The S.D.s of triplicate measurements of Ct were used for error propagation. When a given protein concentration was measured in more than two experiments, ΔCt was averaged across experiments and the average errors calculated.
Adams B.M., Coates M.N., Jackson S.R., Jurica M.S, & Davis T.L. (2015). Nuclear cyclophilins affect spliceosome assembly and function in vitro. The Biochemical journal, 469(2), 223-233.
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2

Quantifying Brain tPA, Nsp, and PAI-1

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RNA was isolated from PBS-perfused WT naïve mouse brains using QIAzol lysis buffer with the RNeasy kit (Qiagen) following manufactured instructions. Quantitative RT-PCR analysis was performed using TaqMan® One-Step RT-PCR Kit and TaqMan® Gene Expression Assays according to Applied Biosystems instructions. FAM-labeled primers for tPA (PLAT; Mm00476931_m1), Nsp (SERPINI1; Mm00436740_m1) and PAI-1 (SERPINE1; Mm00435858_m1) were obtained from ThermoFisher. Fold Change gene expression was expressed relative to the housekeeping gene Beta-Actin (ACTB; Mm00607939_s1) and corrected for primer efficiencies (tPA: 2.08; Nsp: 1.98; PAI-1: 1.91).
Torrente D., Joseph Su E., Fredriksson L., Warnock M., Bushart D., Mann K., Emal C.D, & Lawrence D.A. (2022). Compartmentalized actions of the plasminogen activator inhibitors, PAI-1 and Nsp in ischemic stroke. Translational stroke research, 13(5), 801-815.
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3

Quantitative RT-PCR from Mouse Colon Tissue

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Total RNA from frozen mouse colon tissue was isolated using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using the StepOnePlus detection system (Applied Biosystems, Foster City, CA, USA) and the TaqMan One-Step RT-PCR kit containing AmpliTaq Gold® DNA polymerase [23 (link)]. Specific primers and probes were obtained from Applied Biosystems. Fold changes in qPCR expression were calculated by the 2(−ΔΔCT) method [24 (link)].
Singh S.P., Chand H.S., Banerjee S., Agarwal H., Raizada V., Roy S, & Sopori M. (2019). Acetylcholinesterase Inhibitor Pyridostigmine Bromide Attenuates Gut Pathology and Bacterial Dysbiosis in a Murine Model of Ulcerative Colitis. Digestive Diseases and Sciences, 65(1), 141-149.
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4

RT-qPCR Analysis of Mouse Genes

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Mouse 18S or Gapdh was used as an internal control by RT‐qPCR. The qPCR primers and probes of Gapdh, Egr1, Cyp27b1, Cyp24a1, Hamp, Bmp6, Tfrc, Col1a1, IL6, Fgf23, and 18S were purchased as pre‐optimized reagents (Applied Biosystems/Life Technologies, Inc.) and the TaqMan One‐Step RT‐PCR kit was used to perform qPCR. PCR conditions for all experiments were: 30 min 48°C, 10 min 95°C, followed by 40 cycles of 15 s 95°C and 1 min 60°C. The data was collected and analyzed by a StepOne Plus system (Applied Biosystems/Life Technologies, Inc.).
Liesen M.P., Noonan M.L., Ni P., Agoro R., Hum J.M., Clinkenbeard E.L., Damrath J.G., Wallace J.M., Swallow E.A., Allen M.R, & White K.E. (2022). Segregating the effects of ferric citrate‐mediated iron utilization and FGF23 in a mouse model of CKD. Physiological Reports, 10(11), e15307.
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5

Detection of PEDV RNA in Fecal and Intestinal Samples

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The fecal swab and feces samples were vortexed for 2 min and centrifuged at 3000g for 5 min. For the intestinal samples, 1 g of the tissue was homogenized in 10 mL of phosphate buffer saline (PBS) and centrifuged at 3000g for 10 min. Viral RNA was extracted from 140 µL of the supernatants using the QIAamp Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions and eluted in 60 µL buffer AVE (Qiagen). PEDV RNA was detected using RT-PCR targeting the conserved regions of the PEDV nucleocapsid (N) protein gene as described by Kim et al. (2007) (link), with modifications to fit the ABI StepOne system. Briefly, using the TaqMan One-Step RT-PCR Kit (Applied Biosystems), RT-PCR was performed in a 25 µL reaction mix containing 4 µL RNA, 1 × RT-PCR enzyme mix, 1 × RT-PCR buffer, 40 pmol forward primer (5′-CGCAAAGACTGAACCCACTAA TTT-3′), 40 pmol reverse primer (5′-TTGCCTCTGTTGTTACTTGGAGAT-3′) and 12 pmol probe (Cy5-TGTT GCCATTGCCACGACTCCTGC-BHQ3). Amplification parameters were as follows: an initial step of 10 min at 50 °C, 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 20 s at 60 °C.
Zeng Z., Li T.T., Jin X., Peng F.H., Song N.H., Peng G.Q, & Ge X.Y. (2017). Coexistence of multiple genotypes of porcine epidemic diarrhea virus with novel mutant S genes in the Hubei Province of China in 2016. Virologica Sinica, 32(4), 298-306.
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6

Quantifying HPeV3 Neutralization by mAb

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100 CCID50 of the wild-type (wt) HPeV3 A308/99 prototype strain or the AT12-015 MAR HPeV3 variant were preincubated with serially diluted concentrations of the AT12-015 mAb at 37 °C, 5% CO2 for 1 hour and used to inoculate monolayers of HT29 cells in 96-well microtiter plates in duplicates. At day 7 post-inoculation, 25 µl of the culture supernatants were lysed by addition of 25 µl of Cells-to-cDNA™ II Cell Lysis Buffer (ThermoFisher Scientific, Waltham, MA) and incubated at +72 °C for 15 minutes. HPeV genomic RNA was quantified from 5 µl of each lysed sample using detection primers and a probe described previously47 (link) and a TaqMan One-Step RT-PCR kit (Applied Biosystems). Copy numbers of HPeV genomes in the medium samples were quantified using a standard curve, which was generated by performing the RT-PCR on serial dilutions of a previously constructed control plasmid containing an HPeV amplicon47 (link). Half maximal inhibitory concentrations (IC50) were determined by nonlinear regression analysis performed in GraphPad Prism 7.
Karelehto E., van der Sanden S., Geraets J.A., Domanska A., van der Linden L., Hoogendoorn D., Koen G., van Eijk H., Shakeel S., Beaumont T., de Jong M., Pajkrt D., Butcher S.J, & Wolthers K.C. (2017). Strain-dependent neutralization reveals antigenic variation of human parechovirus 3. Scientific Reports, 7, 12075.
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7

Quantitative Expression Analysis of LO Genes

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Total RNAs were isolated from mouse leukocytes purified from the pooled blood of either five WT or 12/15-LO KO mice using QIAGEN spin-column kits. Subsequently, they subjected to a 50 cycle PCR amplification using TaqMan one-step RT-PCR kit (#4392938) obtained from (Applied Biosystems (ABI), Foster City, CA) with pre-designed TaqMan primers (Catalog#: 12-LO: Mm00545833_m1; 15-LO: Mm00507789_m1; 18S: Mm03928990_g1). 18S was used as the endogenous reference gene.
Ibrahim A.S., Saleh H., El-Shafey M., Hussein K.A., El-Masry K., Baban B., Sheibani N., Wang M.H., Tawfik A, & Al-Shabrawey M. (2017). Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis. Biochimica et biophysica acta, 1862(6), 636-645.
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8

Quantifying Gene Expression Using RT-qPCR

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Kidney and cell RNAs were extracted using Trizol (Life Technologies, Carlsbad, CA) or Lysis buffer (Bioline) for in vivo and in vitro isolation, respectively, according to the manufacturer’s protocols. Samples were tested with primers purchased from Applied Biosystems/Life Technologies, Inc., specific for human or mouse EGR1, CYP27B1, CYP24A1, KL, Hbegf, β-actin and Gapdh. The TaqMan One-Step RT-PCR kit (Life Technologies) was used for all analyses, and data were collected with the 7500 Real Time PCR/StepOne Plus system and software (Life Technologies). The qPCR data was analyzed using the 2−ΔΔCT method according to Livac43 (link).
Ni P., Clinkenbeard E.L., Noonan M.L., Richardville J.M., McClintick J., Hato T., Janosevic D., Cheng Y.H., El-Achkar T.M., Eadon M.T., Dagher P.C, & White K.E. (2020). Targeting fibroblast growth factor 23-responsive pathways uncovers controlling genes in kidney mineral metabolism.. Kidney international, 99(3), 598-608.
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9

Quantitative Real-Time PCR Analysis

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Quantitative real-time PCR (QRTPCR) was performed using the TaqMan One-Step RT-PCR Kit for one step reactions using the 7900 HT RT-PCR system (Life Technologies, Grand Island, NY) with TaqMan probes (Life Technologies, Grand Island, NY). A list of primers and probes is provided in the Supplementary Table 2.
Detailed description of Materials and Methods can be found in the Supplement.
Huntzicker E.G., Hötzel K., Choy L., Che L., Ross J., Pau G., Sharma N., Siebel C.W., Chen X, & French D.M. (2015). Differential effects of targeting Notch receptors in a mouse model of liver cancer. Hepatology (Baltimore, Md.), 61(3), 942-952.
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10

SARS-CoV-2 Quantification via Real-Time RT-PCR

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In this study, real-time RT-PCR was performed to quantify the SARS-CoV-2 level. This PCR amplified the envelope (E) gene of SARS-CoV-2 using the forward primer 5′-ACAGGTACGTTAATAGTTAATAGC-3′; reverse primer: 5′-ATATTGCAGCAGTACGCA-CAC-3′; and probe: 5′-FAMACACTAGCCATCCTTACTGCGCTTCGBBQ-3′. Real-time RT-PCR assays were conducted using a TaqMan One-Step RT-PCR kit (Thermo Fisher Scientific) on a Real-Time PCR System (Bio-Rad, Hercules, CA, USA) with the following cycling conditions: 55°C for 10 min for reverse transcription, 95°C for 3 min, and 45 cycles of 95°C for 15 s and 58°C for 30 s. The absolute copy number of viral loads was determined using serially diluted DNA control targeting the E gene of SARS-CoV-2.
Tran T.N., May B.P., Ung T.T., Nguyen M.K., Nguyen T.T., Dinh V.L., Doan C.C., Tran T.V., Khong H., Nguyen T.T., Hua H.Q., Nguyen V.A., Ha T.P., Phan D.L., Nguyen T.A., Bui T.N., Tu T.M., Nguyen T.T., Le T.T., Dong T.L., Huynh T.H., Ho P.H., Le N.T., Truong C.T., Pham H.P., Luong C.Y., Y N.L., Cao M.N., Nguyen D.K., Le T.T., Vuong D.C., Nguyen L.K, & Do M.S. (2021). Preclinical Immune Response and Safety Evaluation of the Protein Subunit Vaccine Nanocovax for COVID-19. Frontiers in Immunology, 12, 766112.
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