Taqman reverse transcription reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, China, Netherlands, Switzerland, Japan, Australia

TaqMan Reverse Transcription Reagents are a set of reagents used for the conversion of RNA into complementary DNA (cDNA). The reagents include a reverse transcriptase enzyme, random hexamers, and other necessary components for the reverse transcription process.

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971 protocols using taqman reverse transcription reagent

Quantitative RT-PCR for Gene Expression

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RNA was isolated using the miRNEasy Mini Kit (QIAGEN, Cat# 217004) according to the manufacturer’s instructions including on-column DNase treatment (QIAGEN, Cat# 79254). The total RNA (1 µg) was reverse transcribed using TaqMan™ Reverse Transcription Reagents (Invitrogen™, Cat# N8080234). qRT-PCR was conducted using the PowerUp™ SYBR™ Green Master mix (Applied Biosystems, Cat# A25742) according to the manufacturer’s instructions. Primers used include the following: GAPDH Forward 5′-ACATCATCCCTGCCTCTACT-3′, Reverse 5′-TCCACCACTGACACGTTG-3′; S100A4 Forward 5′-CAGAACTAAAGGAGCTGCTGACC-3′, Reverse 5′-CTTGGAAGTCCACCTCGTTGTC-3’ Reactions were run on a QuantStudio 3 (Applied Biosystems) thermocycler. The level of gene expression was calculated by the 2^–ΔΔCT method and normalized to the Ct value for GAPDH.

Rajurkar M., Parikh A.R., Solovyov A., You E., Kulkarni A.S., Chu C., Xu K.H., Jaicks C., Taylor M.S., Wu C., Alexander K.A., Good C.R., Szabolcs A., Gerstberger S., Tran A.V., Xu N., Ebright R.Y., Van Seventer E.E., Vo K.D., Tai E.C., Lu C., Joseph-Chazan J., Raabe M.J., Nieman L.T., Desai N., Arora K.S., Ligorio M., Thapar V., Cohen L., Garden P.M., Senussi Y., Zheng H., Allen J.N., Blaszkowsky L.S., Clark J.W., Goyal L., Wo J.Y., Ryan D.P., Corcoran R.B., Deshpande V., Rivera M.N., Aryee M.J., Hong T.S., Berger S.L., Walt D.R., Burns K.H., Park P.J., Greenbaum B.D, & Ting D.T. (2022). Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer. Cancer discovery, 12(6), 1462-1481.

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Gene Expression Analysis via RT-qPCR

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RT reactions were performed using TaqMan Reverse Transcription Reagents (Invitrogen, N8080234) following the manufacturer’s protocol. All qPCRs were carried out on the Agilent M×3000P Real-Time PCR System with FastStart Universal SYBR Green Master Mix (Roche, 4913914001) following the standard protocol. Gene expression was normalized to the expression of the PtrACTIN gene. The primers used for RT–qPCR and ChIP–qPCR are listed in Supplementary Table 6.

Dai X., Zhai R., Lin J., Wang Z., Meng D., Li M., Mao Y., Gao B., Ma H., Zhang B., Sun Y., Li S., Zhou C., Lin Y.C., Wang J.P., Chiang V.L, & Li W. (2023). Cell-type-specific PtrWOX4a and PtrVCS2 form a regulatory nexus with a histone modification system for stem cambium development in Populus trichocarpa. Nature Plants, 9(1), 96-111.

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Quantitative RT-PCR analysis of BDNF

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Total RNA was extracted from HEK293 cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany 74106) with on-column performed deoxyribonuclease (DNase) treatment using a Ribonuclease-Free DNase Kit (Qiagen), according to the manufacturer’s protocol. The quality of RNAs was checked using NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA (1 µg) was then reverse-transcribed using TaqMan reverse transcription reagents (Invitrogen, Cat No. N8080234). Gene expression was then measured by real-time PCR (RT-PCR) using human BDNF (Invitrogen, cat No. 444889, assay ID Hs02718934_s1) and human ACTB (β-actin) endogenous control (Invitrogen, cat No. 4326315E) TaqMan primer-probe assays and TaqMan gene expression master mix (Invitrogen 4369016). Samples were amplified for 40 cycles using the Applied Biosystem Quantstudio Flex 6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative gene expression presented was calculated based on fold change using the delta C_t method. Data were analyzed using two-tailed Student’s t test with GraphPad Prism software (San Diego, CA, USA).

Bagheri A., Habibzadeh P., Razavipour S.F., Volmar C.H., Chee N.T., Brothers S.P., Wahlestedt C., Mowla S.J, & Faghihi M.A. (2019). HDAC Inhibitors Induce BDNF Expression and Promote Neurite Outgrowth in Human Neural Progenitor Cells-Derived Neurons. International Journal of Molecular Sciences, 20(5), 1109.

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Quantitative RNA Expression Analysis

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RNA was collected (Qiagen, RNeasy) and reverse transcribed to complementary DNA (cDNA) using the TaqMan Reverse Transcription Reagents (Invitrogen) using the Oligo d(T) method. cDNA was then used as template for quantitative real time PCR with specific human primers using Power SYBR Green PCR master mixes (Thermo Fisher) using a ViiA 7 real-time PCR system (Applied Bio-systems). Target expression was normalized to β2M or 18S where indicated and relative expression was calculated using the delta-delta CT method. For qPCR timecourses, data is normalized to the respective first collected control time point (either 4 or 24 h after dexamethasone synchronization).

Brooks R., Monzy J., Aaron B., Zhang X., Kossenkov A., Hayden J., Keeney F., Speicher D.W., Zhang L, & Dang C.V. (2022). Circadian lncRNA ADIRF-AS1 binds PBAF and regulates renal clear cell tumorigenesis. Cell reports, 41(3), 111514.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). Complementary DNA was generated using the TaqMan Reverse Transcription Reagents (Invitrogen). qPCR was performed in triplicate or quadruplicate using the Power SYBR Green PCR Master Mix (Invitrogen) on a 7500 Real-Time PCR System (Applied Biosystems) or a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Specificity of amplification was confirmed by melting curve analysis. Cycle threshold (C_T) values for target genes were normalized to the endogenous reference gene GAPDH. Primer sequences (Table S3) were designed from the University of California Santa Cruz genome browser reference mRNA sequences using Primer3 (43 ).

Heppler L.N., Attarha S., Persaud R., Brown J.I., Wang P., Petrova B., Tošić I., Burton F.B., Flamand Y., Walker S.R., Yeh J.E., Zubarev R.A., Gaetani M., Kanarek N., Page B.D, & Frank D.A. (2021). The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase. The Journal of Biological Chemistry, 298(2), 101531.

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Quantitative Analysis of Chondrocyte Markers

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Total RNA was extracted from monolayer or pellet cultures and oligo(dT)-primed cDNA was prepared using the TaqMan® Reverse Transcription Reagents (Invitrogen). The levels of EFEMP1, Sox9, ACAN, Col2a1, and GAPDH gene expression were assessed by using Taqman primers and probes (Applied Biosystems) and normalized to GAPDH.

Hasegawa A., Yonezawa T., Taniguchi N., Otabe K., Akasaki Y., Matsukawa T., Saito M., Neo M., Marmorstein L.Y, & Lotz M.K. (2017). Fibulin-3 in joint aging and osteoarthritis pathogenesis. Arthritis & rheumatology (Hoboken, N.J.), 69(3), 576-585.

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Circular RNA and miRNA Expression Analysis

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Tissues or cells were lysed using Trizol reagent (Invitrogen) to obtain total RNA. Complementary DNA (cDNA) was synthesized from RNA using TaqMan Reverse Transcription Reagents (Invitrogen) or MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Afterwards, cDNA was amplified for qPCR using Fast SYBR Green Master Mix (Applied Biosystems) through a BioRad CFX96 system (Bio-Rad, Hercules, CA, USA). Relative expression was calculated by the 2^Ct method, with GAPDH or U6 as an internal reference. The primers used were listed as below: circ_0000745, F: 5-GGCCAAGGGGCCTTTACAA-3 and R: 5-GTGGCACAGACCTCTCTCTT-3; miR-488, F: 5-TGCGGCTTGAAAGGCTATT-3 and R: 5-ATGGAGCCTGGGACGAGAC-3; U6, F: 5-CTCGCTTCGGCAGCACA-3 and R: 5-AACGCTTCACGAATTTGCGT-3; GAPDH, F: 5-GCACCGTCAAGGCTGAGAAC-3 and R: 5-TGGTGAAGACGCCAGTGGA-3; CCND1, F: 5-AGCTGTGCATCTACACCGAC-3 and R: 5-GAAATCGTGCGGGGTCATTG-3.

Li K., Fan X., Yan Z., Zhan J., Cao F, & Jiang Y. (2021). Circ_0000745 strengthens the expression of CCND1 by functioning as miR-488 sponge and interacting with HuR binding protein to facilitate the development of oral squamous cell carcinoma. Cancer Cell International, 21, 271.

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Quantifying mRNA Expression in Fibroblasts

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RNA from in vitro fibroblast samples and in vivo tissue samples was collected then phase separated using Trizol (Invitrogen) and isolated using the RNeasy Mini Kit (Qiagen). Isolated RNA was quantified using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA) then reverse-transcribed using TaqMan Reverse Transcription Reagents (Invitrogen). cDNA was examined using quantitative real-time PCR (qRT-PCR) with the Applied Biosystems Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA) and Power SYBR Green PCR Master Mix (Applied Biosystems). Target quantities were normalized to endogenous β-actin quantities using the standard curve method. Normalized quantities were then calibrated to baseline expression of shScr to generate relative expression levels. Primer sequences were obtained from PrimerBank (S1 Table).

Paik K.J., Maan Z.N., Zielins E.R., Duscher D., Whittam A.J., Morrison S.D., Brett E.A., Ransom R.C., Hu M.S., Wu J.C., Gurtner G.C., Longaker M.T, & Wan D.C. (2016). Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs. PLoS ONE, 11(3), e0150927.

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Optic Nerve RNA Extraction and qRT-PCR

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Optic nerves were removed from mice after transcardiac perfusion with cold, sterile PBS, and RNA was extracted using the RNeasy Mini Kit (74104, QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration was measured using a NanoDrop One (Thermo Fisher Scientific, Waltham, MA), and any remaining DNA contamination was removed by treatment with deoxyribonuclease I (18068015, Invitrogen, Carlsbad, CA). The complementary DNA was synthesized using TaqMan Reverse Transcription Reagents (N8080234, Invitrogen, Carlsbad, CA). Quantitative real-time reverse transcription polymerase chain reactions (PCR) were made using TaqMan Fast Advanced Master Mix (4444556, Applied Biosystems, Foster City, CA) and predeveloped TaqMan gene expression probes. Reactions were analyzed on a QuantStudio 6 Flex Real-Time PCR System using QuantStudio 6 software (Applied Biosystems, Foster City, CA). Samples were run in triplicate for each gene, and data were averaged for analysis. Fold change was calculated as 2^−ΔΔCt compared to PLPcreER⁺;GluA4^fl/fl (WT) values. Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase.

Evonuk K.S., Doyle R.E., Moseley C.E., Thornell I.M., Adler K., Bingaman A.M., Bevensee M.O., Weaver C.T., Min B, & DeSilva T.M. (2020). Reduction of AMPA receptor activity on mature oligodendrocytes attenuates loss of myelinated axons in autoimmune neuroinflammation. Science Advances, 6(2), eaax5936.

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RNA Isolation and qPCR Analysis

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Total RNA was isolated using the RNeasy Mini Kit (Qiagen), treated with TURBO DNase to remove potential genomic DNA contamination and reverse transcribed into cDNA using the TaqMan Reverse Transcription Reagents (Invitrogen). PCR amplification was carried out with GoTaq Master Mix (Promega) and specific forward and reverse primers. The PCR products were analyzed by electrophoresis with 1% agarose gel, stained by ethidium bromide and visualized under UV light. Further detail is provided in the Supplementary Information.

Yang L., Li Y, & Zhang Y. (2014). Identification of prolidase as a high affinity ligand of the ErbB2 receptor and its regulation of ErbB2 signaling and cell growth. Cell Death & Disease, 5(5), e1211-.

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