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Taqman reverse transcriptase

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The TaqMan Reverse Transcriptase is a thermostable enzyme used for the reverse transcription of RNA to cDNA in molecular biology applications. It catalyzes the synthesis of first-strand cDNA from RNA templates.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (6 mentions) Rneasy mini kit, by Qiagen (3 mentions) Power sybr green, by Thermo Fisher Scientific (3 mentions) Abi prism 7700 sequence detector, by Thermo Fisher Scientific (2 mentions) 7500 real time polymerase chain reaction system, by Thermo Fisher Scientific (1 mentions) Smart cycler 2, by Cepheid (1 mentions) Mirvana mirna extraction kit, by Thermo Fisher Scientific (1 mentions) Abi prism 7000 pcr cycler, by Thermo Fisher Scientific (1 mentions) Cfx96 real time pcr detection system, by Bio-Rad (1 mentions) Amplitaq, by Thermo Fisher Scientific (1 mentions) Random hexamer primer, by Roche (1 mentions) Magna pure lc total nucleic acid isolation kit, by Roche (1 mentions) Rq1 rnase free dnase, by Promega (1 mentions) Faststart sybr green master, by Roche (1 mentions) Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Rneasy kit, by Qiagen (1 mentions) Abi prism 7900 detection system, by Thermo Fisher Scientific (1 mentions) Abi prism 7700, by Thermo Fisher Scientific (1 mentions) Tri reagent, by Merck Group (1 mentions) Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)

12 protocols using taqman reverse transcriptase

1

Quantitative RT-PCR for Osteogenic Markers

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Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and cleaned with an RNeasy Mini Kit (Qiagen). Single strand cDNA was synthesized using Taqman reverse transcriptase (Applied Biosystems). Quantitative real-time polymerase chain reaction (PCR) measurement of Bglap2, Ibsp, and Gapdh mRNA was carried out as described previously(22) using predeveloped Taqman probes and an ABI PRISM 7700 sequence detector (Applied Biosystems). mRNA expression was normalized to Gapdh mRNA.
Li Y., Ge C, & Franceschi R.T. (2017). MAP Kinase-dependent RUNX2 Phosphorylation is Necessary for Epigenetic Modification of Chromatin During Osteoblast Differentiation. Journal of cellular physiology, 232(9), 2427-2435.
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2

Quantitative Real-Time PCR Analysis of Cytokine Expression

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Total RNA from the post-ischemic hemisphere was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, United States) according to the manufacturer’s instructions. cDNA was obtained using Taqman reverse trans-criptase (Applied Biosystems; Thermo Fisher Scientific, Inc.). TGF-β and β-actin cDNA were amplified using Power SYBR Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). Two-step qPCR was performed (95°C for 15 s, 60°C for 60 s for 40 cycles) with specific primers for IL-6 (forward, 5′-GAGGATACCACTCCCAACAGACC-3′; reverse: 5′-GAGGGATATCTATCAGG GTCTTCAT-3′), IL-10 (forward, 5′–3′ and reverse, 5′–3′), TGF-β (forward, 5′-GTGTGGAGCAACATGTGGAACTCTA-3′ and reverse, 5′-TTGGTTCAGCCACTGCCGTA-3′), IL-4 (for-ward, 5′-ACCAGGAGCCATATCCAC-3′; reverse: 5′-TTGGAAGCCCTACAGACG-3′), IFN-γ (forward, 5′-TCAAGTGGCATAGATGTGGAAGA-3′ and reverse, 5′-GAGATAATCTGGCTCTGCAGGATT-3′) and β-actin (forward, 5′-AAGGCCAACCGTGAAAAGAT-3′ and reverse, 5′-GTGGTACGACCAGAGGCATAC-3′). The relative quantitation value is expressed as 2-ΔΔCq, where ΔCq is the difference between the mean ΔCq value of duplicate measurements of the sample and β-actin control.
Yao H., Zhang Y., Shu H., Xie B., Tao Y., Yuan Y., Shang Y., Yuan S, & Zhang J. (2019). Hyperforin Promotes Post-stroke Neuroangiogenesis via Astrocytic IL-6-Mediated Negative Immune Regulation in the Ischemic Brain. Frontiers in Cellular Neuroscience, 13, 201.
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3

Quantitative PCR of Stem Cell Markers

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Total RNA was isolated from epithelial cell cultures or prostate tissue using TRIzol reagent (Invitrogen) using an RNeasy Mini Kit (Qiagen). For cDNA synthesis, 2 μg of total RNA was reverse-transcribed using TaqMan reverse transcriptase (Applied Biosystems, Inc.). The following mRNAs were detected by real-time polymerase chain reaction (qPCR) using TaqMan probes and an ABI PRISM 7700 sequence detector (Applied Biosystems): Runx1, Runx2, Runx3, Bmi1, Nanog, Sox2, Cd44, Prom1 (CD133), Krt5, Krt8, Syn, Psca, Pbsn and Sbp. The mRNA expression for each gene was calculated based on a relative standard and normalized to Gapdh mRNA.
Li Y., Ge C, & Franceschi R.T. (2021). Role of Runx2 in Prostate Development and Stem Cell Function. The Prostate, 81(4), 231-241.
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4

Quantitative RT-PCR Analysis of TGF-β

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Total RNA from the post-ischemic hemisphere was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. cDNA was obtained using Taqman reverse transcriptase (Applied Biosystems; Thermo Fisher Scientific, Inc.). TGF-β and β-actin cDNA were amplified using Power SYBR-Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). Two-step qPCR was performed (95°C for 15 sec, 60°C for 60 sec for 40 cycles) with specific primers for TGF-β (forward, 5′-GTGTGGAGCAACATGTGGAACTCTA-3′ and reverse, 5′-TTGGTTCAGCCACTGCCGTA-3′) and β-actin (forward, 5′-AAGGCCAACCGTGAAAAGAT-3′ and reverse, 5′-GTGGTACGACCAGAGGCATAC-3′). The relative quantitation value is expressed as 2−ΔΔCq, where ΔCq is the difference between the mean ΔCq value of duplicate measurements of the sample and β-actin control (30 (link)).
Zhang Y., Yu P., Liu H., Yao H., Yao S., Yuan S.Y, & Zhang J.C. (2018). Hyperforin improves post-stroke social isolation-induced exaggeration of PSD and PSA via TGF-β. International Journal of Molecular Medicine, 43(1), 413-425.
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5

Quantitative Analysis of iNOS and β-actin mRNA

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Real-time RT-PCR was used to assess abundances of iNOS and β-actin mRNA. Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total RNA with Taqman reverse transcriptase (Applied Biosystems, Foster City, CA, USA). cDNA were amplified in a Smart Cycler II (Cepheid, Sunnyvale, CA, USA) by a SYBR Green Polymerase Chain Reaction Master Mix (Promega, Madison, WI, USA) and an Applied Biosystems 7500 Real-Time Polymerase Chain Reaction System (Carlsbad, CA, USA). Two-step real-time polymerase chain reaction was performed (95°C for 15 seconds, 60°C for 60 sec extension and detection, 40 cycles) with specific primers for iNOS (forward 5′-CTTTTAGAGACGCTTCTGA G-3′ and reverse 5′-TTTGATGCTTGTGACTCTTA-3′) and β-actin (forward: 5′-TCTTTTCCAG CCTTCCTTCTTG-3′; reverse: 5′-GCACTGTGTTGGCATAGAGGTC-3′). Abundances of amplified genes were assessed by analysis of cycle threshold.
Si J., Wang N., Wang H., Xie J., Yang J., Yi H., Shi Z., Ma J., Wang W., Yang L., Yu S, & Li J. (2014). HIF-1α Signaling Activation by Post-Ischemia Treatment with Astragaloside IV Attenuates Myocardial Ischemia-Reperfusion Injury. PLoS ONE, 9(9), e107832.
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6

Quantifying Gene Expression by qRT-PCR

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After 6 hr stimulation, total RNA was isolated from the cell lysate using the miRVana miRNA Extraction kit (Ambion) according to the instructions of the manufacturer. cDNA was synthesized with TaqMan Reverse Transcriptase (Applied Biosystems, Foster City, CA) and mRNA expression of genes were determined by qRT-PCR. Real-time qRT-PCR was performed on an ABI-Prism 7000 PCR cycler (Applied Biosystems) or on the CFX96 Real-Time PCR Detection System (Bio-Rad). Cycling parameters were 95°C for 1 min and then 35 cycles of 95°C (15 s) and 60°C (1 min), followed by a melting curve analysis. The median cycle threshold (Ct) value and 2-ΔΔCt method were used for relative quantification analysis, and all Ct values were normalized to the GAPDH mRNA expression level. Results expressed as means and SEM of biological replicates are shown. The mock sample (HUVECs incubated with culture media only) was used as reference. The oligonucleotides used are described in Supplementary file 2.
Silva-Filho J.L., Dos-Santos J.C., Judice C., Beraldi D., Venugopal K., Lima D., Nakaya H.I., De Paula E.V., Lopes S.C., Lacerda M.V., Marti M, & Costa F.T. (2021). Total parasite biomass but not peripheral parasitaemia is associated with endothelial and haematological perturbations in Plasmodium vivax patients. eLife, 10, e71351.
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7

FMDV RNA Quantification in Biological Samples

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Total RNA was extracted from serum, nasal fluid, and OPF samples using a MagNA Pure LC total nucleic acid isolation kit (product no. 03038505001; Roche) with an automated robotic workstation (Roche), according to the manufacturer’s instructions. The level of FMDV RNA in serum and nasal fluid samples was determined using a one-step quantitative RT-PCR assay targeting the 3D-coding region of the FMDV genome (52 (link)) as modified by Vandenbussche et al. (53 (link)). OPF samples were analyzed using similar primers and probes, but in a two-step assay. Briefly, cDNA was produced using reverse transcriptase (TaqMan reverse transcriptase; Applied Biosystems) with random hexamer primers (Roche). The quantitative PCR was performed using AmpliTaq (Applied Biosystems) using the primers and probes described by Callahan et al. (52 (link)).
Arzt J., Belsham G.J., Lohse L., Bøtner A, & Stenfeldt C. (2018). Transmission of Foot-and-Mouth Disease from Persistently Infected Carrier Cattle to Naive Cattle via Transfer of Oropharyngeal Fluid. mSphere, 3(5), e00365-18.
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8

Silkworm Immune Response to P. aeruginosa

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Silkworms on day 1 of the fifth instar stage were fed an artificial diet containing heat-killed P. aeruginosa cells and incubated for 2 d at 27°C. Total RNA from hemocytes, midgut, and fat body of the silkworms was extracted using an RNeasy Mini Kit (Qiagen), according to the manufacturer’s protocol. After digestion of the contaminated genomic DNA by RQ1 RNase-free DNase (Promega), the RNA was reverse-transcribed to cDNA using TaqMan reverse transcriptase (Applied Biosystems). Quantitative real-time polymerase chain reaction was performed using oligonucleotide primers (Table 1), cDNAs as a template, and FastStart SYBR Green Master (Roche).
Miyashita A., Takahashi S., Ishii K., Sekimizu K, & Kaito C. (2015). Primed Immune Responses Triggered by Ingested Bacteria Lead to Systemic Infection Tolerance in Silkworms. PLoS ONE, 10(6), e0130486.
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9

Quantitative PCR Analysis of Prefrontal Cortex in Mice

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The prefrontal cortex was dissected from 2-month-old mice and quickly frozen at −80 °C. RNA was extracted with an RNeasy kit (Qiagen), and complementary DNA was synthesized with random hexamers and TaqMan reverse transcriptase (Applied Biosystems). For quantitative PCR, primers specific for Flag-(GR)80 are: forward primer 5′-GGCGCTTTCTCATAG CTCAC-3′ and reverse primer 5′-GCCTACATACCTCGCTCTGC-3′. Primers for Atp5a1 are: forward primer 5′-GCCCTCGGTAATGCTATTGA-3′ and reverse primer 5’-GCAATCGATG TTTTCCCAGT-3′. For quantitative PCR, these primers were used in reactions with SYBR Green PCR master mix (Applied Biosystems). Gapdh-specific primers were used as an internal control: forward primer 5′-AACTTTGGCATTGTGGAAGG-3′ and reverse primer 5′-ACACATTGGG GGTAGGAACA-3′.
Choi S.Y., Lopez-Gonzalez R., Krishnan G., Phillips H.L., Li A.N., Seeley W.W., Yao W.D., Almeida S, & Gao F.B. (2019). C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo. Nature neuroscience, 22(6), 851-862.
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10

Gene Expression Analysis of Mechano-Sensitive Ion Channels

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RNA was extracted from cells using TriReagent (Sigma-Aldrich) and isolated, followed by cDNA synthesis using TaqMan reverse transcriptase (Applied Biosystems). cDNA samples were amplified with Trpv4 (Mm00499025_m1), Pkd2 (Mm00435829_m1), Piezo1 (Mm01241570_g1), Piezo2 (Mm01262433_m1), Cox-2 (Mm00478374_m1), and Gapdh (4352339E) primers and probes (Applied Biosystems) by quantitative real-time RT-PCR using the ABI PRISM 7900 detection system (Applied Biosystems). Samples and standards were run in triplicate and were normalized to the endogenous Gapdh expression. Relative gene levels between samples were determined using the relative standard curve method (ABI Prism 7700 User Bulletin 2; Applied Biosystems).
Lee K.L., Guevarra M.D., Nguyen A.M., Chua M.C., Wang Y, & Jacobs C.R. (2015). The primary cilium functions as a mechanical and calcium signaling nexus. Cilia, 4, 7.
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