Taqman microrna rt kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Canada, Japan, United Kingdom

The TaqMan MicroRNA RT Kit is a reagent kit designed for reverse transcription of mature microRNA (miRNA) molecules. The kit includes the necessary components to perform reverse transcription of miRNA samples, allowing for the subsequent quantification of miRNA expression levels using real-time PCR techniques.

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TaqMan kits (18 citations) TaqMan microRNA kit (15 citations) MicroRNA RT kit (9 citations) RT kits (5 citations) TAQMAN(R) MICRORNA RT KIT (2 citations)

165 protocols using taqman microrna rt kit

Quantitative Analysis of miRNA and mRNA

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Total miRNA was extracted using miRNApure Mini Kit (CWBiotech), according to the manufacturer's instruction. Reverse transcription was performed using Taqman microRNA RT Kit (Life Technologies) and Taqman microRNA Assay with specific stem-loop primers (Life Technologies). Real-time PCR was performed using Taqman Universal Master Mix II (Life Technologies). The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min using ABI 7500 real-time PCR system. Results were normalized to the internal control, RNU6B.
Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer's instruction. Reverse transcription reactions were performed using High Capacity RNA-to-cDNA Kit (Life Technologies). Real-time PCR was performed in ABI 7500 real-time PCR system using SYBR Green PCR Master Mix (Life Technologies). The primers used are shown in Supplementary Table S1. The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. GAPDH was used as internal control.
All reverse transcription reactions included no-template controls, and all PCR reactions were run in triplicates. Relative miRNA or mRNA expression was determined using the comparative C_T (2^−ΔΔCt) method.

Yang S., Wu B., Sun H., Ji F., Sun T., Zhao Y, & Zhou D. (2016). Interrupted E2F1-miR-34c-SCF negative feedback loop by hyper-methylation promotes colorectal cancer cell proliferation. Bioscience Reports, 36(1), e00293.

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Exosomal miRNA Quantification Protocol

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RNA was isolated using the seraMir Exosome RNA purification kit (RA806A-1, SBI, Palo Alto, CA, USA). All isolation processes were performed according to the manufacture’s protocol. To quantify EV miRNA markers, the RNA eluate solution was subjected to reverse transcription with the TaqMan MicroRNA RT kit (4366596, Life Technologies, Eugene, OR, USA) and TaqMan Micro RNA Assays (4427975, Life Technologies, USA). TaqMan Universal Master Mix II, no UNG (4440040, Life Technologies, Eugene, OR, USA) was used, together with the miRNA assays hsa-let-7a-5p, ID 000377, and hsa-miR-142-3p, ID 000464. Further experiments were performed with RNA eluate using the Agilent Eukaryote Total RNA Pico chip on an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Kim J., Lee H., Park K, & Shin S. (2020). Rapid and Efficient Isolation of Exosomes by Clustering and Scattering. Journal of Clinical Medicine, 9(3), 650.

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Quantification of Gene and miRNA Expression

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cDNA was generated from 1 μg RNA with SuperscriptII (Invitrogen). qRT-PCR was performed with the LightCycler 480II (Roche, Mannheim, Germany) using the Universal Probe System (Roche). Expression levels were normalized to ribosomal protein L13A (RPL13A), which is a well-proven housekeeping gene in keratinocytes [73 (link)], or beta-actin as described [74 (link)]. Intron-spanning oligonucleotides were designed with the Universal Probe Library (UPL) software (Roche) (S2 Table).
miRNA was transcribed using TaqMan MicroRNA RT Kit (Life technologies) with oligonucleotides for hsa-miR-203 and hsa-RNU6B in each reaction. miRNA-cDNA detection was based on TaqMan MicroRNA Assays (Life Technologies). The amount was calculated by the 2^{–[Δ][Δ]Ct} method normalizing to RNU6B.

Marthaler A.M., Podgorska M., Feld P., Fingerle A., Knerr-Rupp K., Grässer F., Smola H., Roemer K., Ebert E., Kim Y.J., Bohle R.M., Müller C.S., Reichrath J., Vogt T., Malejczyk M., Majewski S, & Smola S. (2017). Identification of C/EBPα as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes. PLoS Pathogens, 13(6), e1006406.

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Quantifying Gene and miRNA Expression in HepG2 Cells

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To determine the mRNA expression levels of PCSK9, PPARγ, and LDLR, total RNA was extracted from HepG2 cells using TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Complementary DNA was synthesized from 2 μg total RNA using a reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). All samples were processed in triplicate using SYBR Green Master Mix (Qiagen, Hilden, Germany) and the ABI7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). Melting curve analysis was performed to confirm the presence of specific PCR products. Gene expression levels were quantified relative to GADPH expression using the comparative Ct (2^−ΔΔCt) value method. The primer sequences used for RT-PCR are listed in Table S1.
For miRNA quantification, 10 ng of total RNA was used for miRNA-specific cDNA synthesis with TaqMan MicroRNA RT kit and quantitative RT-PCR was performed with the TaqMan MicroRNA Assay following the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA). Each reaction was run in triplicate. U6 was detected as the internal control.

Chen P.W., Tseng S.Y., Chang H.Y., Lee C.H, & Chao T.H. (2022). Diverse Effects of Cilostazol on Proprotein Convertase Subtilisin/Kexin Type 9 between Obesity and Non-Obesity. International Journal of Molecular Sciences, 23(17), 9768.

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RNA Extraction and Expression Analysis

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Total RNA was extracted from cells or protein A or G beads with TRIzol (Life Technologies). Reverse transcription was performed using the Superscript III First-Strand Synthesis SuperMix for qRT-PCR Kit (catalog number 11752-050, Life Technologies) for mRNA quantitation or the TaqMan microRNA RT Kit (catalog number 4366596, Life Technologies) for miRNA quantitation. PCR was performed in triplicate on an ABI 7900HT (Applied Biosystems) using the following TaqMan expression assays (Life Technologies): miR-1, 000385; miR-206, 000510; miR-133b, 002247; Igf-1, Mm00439560_m1; and HDAC4, Mm01299558-g1, and was analyzed with SDS software (Life Technologies).

King I.N., Yartseva V., Salas D., Kumar A., Heidersbach A., Ando D.M., Stallings N.R., Elliott J.L., Srivastava D, & Ivey K.N. (2014). The RNA-binding Protein TDP-43 Selectively Disrupts MicroRNA-1/206 Incorporation into the RNA-induced Silencing Complex. The Journal of Biological Chemistry, 289(20), 14263-14271.

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MicroRNA Isolation and Quantification

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MicroRNAs were isolated using Directzol RNA mini-prep kit (Zymo Research). Reverse transcription was performed using TaqMan MicroRNA RT Kit, resulting cDNA underwent pre-amplification with TaqMan PreAmp Master Mix, and RTqPCR was performed with TaqMan MicroRNA assays and TaqMan Universal Master Mix II containing UNG (Life Technologies).

Bogenberger J., Whatcott C., Hansen N., Delman D., Shi C.X., Kim W., Haws H., Soh K., Lee Y.S., Peterson P., Siddiqui-Jain A., Weitman S., Stewart K., Bearss D., Mesa R., Warner S, & Tibes R. (2017). Combined venetoclax and alvocidib in acute myeloid leukemia. Oncotarget, 8(63), 107206-107222.

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Urinary Exosomal miRNA Profiling by qPCR

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Complementary DNA (cDNA) was synthesized from urinary exosomal RNA using the TaqMan MicroRNA RT Kit (Life Technologies) according to the manufacturer’s instructions. Quantitative PCR for mature miRNAs was performed using TaqMan miRNA Gene Assays (Life Technologies) and Gene Expression Master Mix (Life Technologies). Primers for hsa-mir-10b, hsa-mir-223, and hsa-mir-200c were obtained from Life Technologies.

Markowska A., Pendergrast R.S., Pendergrast J.S, & Pendergrast P.S. (2017). A novel method for the isolation of extracellular vesicles and RNA from urine. Journal of Circulating Biomarkers, 6, 1849454417712666.

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Quantification of miR-146a Expression in Chondrocytes

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Total RNA in chondrocytes was extracted and quantified as described (Guan, Yang, Yang, Charbonneau & Chen, 2014; Yang et al., 2016). miR‐146a expression level was quantified with the TaqMan microRNA assays specific for mature miR‐146a (Life Technologies). Briefly, 10 ng RNA was transcribed using TaqMan microRNA RT Kit (Life Technologies) and followed by real‐time PCR with TaqMan MicroRNA Assays. The ubiquitously expressed miRNA, snoRNA U6, was used as an endogenous control. The mRNA levels were quantified by real‐time PCR with the SYBR Green PCR Master Mix (Qiagen). 18S ribosomal RNA was used as an internal control gene to normalize the mRNA levels. Relative transcription levels were calculated as previously described (Guan et al., 2011; Yang et al., 2016). In situ hybridization of miR‐146a was performed according to the manufacturer's instructions. LNA TM detection probe of miR‐146a and a control probe were purchased from EXIQON Inc. (Woburn, MA). Sections were hybridized with 40 nM double‐DIG LNATM miR‐146a Probe for 1 hr at 55°C. Staining was performed using BCIP and nitroblue tetrazolium (Roche, Branchburg, NJ).

Guan Y., Li J., Yang X., Du S., Ding J., Gao Y., Zhang Y., Yang K, & Chen Q. (2018). Evidence that miR‐146a attenuates aging‐ and trauma‐induced osteoarthritis by inhibiting Notch1, IL‐6, and IL‐1 mediated catabolism. Aging Cell, 17(3), e12752.

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Quantitative PCR Analysis of miRNA and mRNA

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Ten nanograms of total RNA was retrotranscribed using TaqMan MicroRNA RT Kit (Life Technologies). Transfected plant miRNA expression levels were measured by TaqMan MicroRNA Assay (Life Technologies) in triplicate, using the ABI PRISM 7900HT platform (Applied Biosystems^®, Life Technologies^TM). snU6 expression was used for RNA normalization (code 001973 Life Technologies). Five hundred nanograms of total RNA were reverse transcribed by using QuantiTect^® Reverse Transcription kit (Qiagen) to measure target genes expression. RT-qPCR experiments were carried out on HCT116 p53^+/+ and HCT116 p53^–/– cell lines to gage target genes expression by using TaqMan^® assays (Life Technologies). The expression levels of target genes were normalized by using the mean expression levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene, selected as the most stable housekeeping gene (between ACTB, GAPDH and RPL13) by the geNorm VBA applet for Microsoft Excel. Negative controls were included without template (NTCs) for each TaqMan assay.
The average of at least three independent experiments was performed and represented in graphs with standard deviation. Student’s t test was used for statistical analysis, and p < 0.05 was considered to be statistically significant.

Marzano F., Caratozzolo M.F., Consiglio A., Licciulli F., Liuni S., Sbisà E., D’Elia D., Tullo A, & Catalano D. (2020). Plant miRNAs Reduce Cancer Cell Proliferation by Targeting MALAT1 and NEAT1: A Beneficial Cross-Kingdom Interaction. Frontiers in Genetics, 11, 552490.

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Reverse Transcription and Profiling of Rodent microRNAs

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Example 3

Reverse transcription (RT) was performed with TaqMan micro RNA RT Kit (Life Technologies, Carlsbad, Calif., USA) as described with slight modifications (Balakathiresan et al., 2012). micro RNA quantity was measured from the total RNA of bioanalyzer data and was used as template RNA (5 ng-brain μRNA; 30 ng-serum μRNA) for RT reactions (FIG. 5). Briefly, the RT reaction mixture contained 0.8 μl Megaplex RT primers Rodent Pool A/B (v3.0), 0.2 μl 100 mM dNTPs (with dTTP), 1.5 μl Multiscribe reverse transcriptase (50 U/μl), 0.8 μl 10×RT Buffer, 0.9 μl MgCl₂(25 mM), 0.1 μl RNAse inhibitor (20 U/μl), RNA template and nuclease free water to a final volume of 7.5 μl. RT reaction was carried out on Veriti 96-Well Thermal Cycler (Life Technologies, Carlsbad, Calif., USA) according to manufacturer's recommended thermal cycling conditions. Pre-amplification of RT products, cycles and conditions were followed according to the manufacturer's protocol (Life Technologies, Carlsbad, Calif., USA). The undiluted pre-amplification products were used for the micro RNA profiling using TaqMan Low Density Rodent microRNAs Array (TLDA) Set v3.0 (Applied Biosystems, Inc) containing 692 rodent micro RNAs. The quantitative PCR (qPCR) reaction was carried out at default thermal-cycling conditions in ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, Life Technologies, Foster City, Calif.).

US10370717B2. Methods for the measurement of post-traumatic stress disorder microRNA markers (2019-08-06). The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. [US]. Inventors: Radha K. Maheshwari [US], Nagaraja S. Balakathiresan [US], Manish Bhomia [US], Raghavendar Chandran [US].

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