Taqman mirna rt kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TaqMan miRNA RT kit is a laboratory tool designed for the reverse transcription of mature microRNA (miRNA) molecules. It enables the conversion of miRNA into cDNA, which can then be used for further analysis and quantification using real-time PCR techniques.

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61 protocols using taqman mirna rt kit

Rodent miRNA RT-qPCR Profiling

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RT was performed with TaqMan miRNA RT kit (Applied Biosystems) as previously described (Nunez et al. 2020 (link)) with slight modifications. Briefly, a RT reaction mixture consisting of 0.8 μl megaplex RT primers (Rodent Pools A + B), 0.2 μl 100 mM dNTPs (with dTTP), 1.5 μl multiscribe reverse transcriptase (50U/μl), 0.8 μl 10X RT buffer, 0.9 μl MgCl2 (25 mM), 0.1 μl RNase inhibitor (20U/μl), 350 ng RNA template, and nuclease-free water to a final volume of 7.5 μl was prepared. RT reaction was carried out on a BioRadT100™ thermal cycler according to the manufacturer’s recommended thermal cycling conditions.
Pre-amplification of the cDNA product after RT was performed using 12.5 µl TaqMan preAmp master mix (2X), 2.5 µl megaplex preAmp primers (Rodent Pools A + B) (10X) and nuclease-free water to a final volume of 25.0 ul in a BioRadT100™ thermal cycler according to the manufacturer’s recommended thermal cycling conditions.

Wijesinghe P., Nunez D.A, & Garnis C. (2022). MicroRNA Signature and Cellular Characterization of Undifferentiated and Differentiated House Ear Institute-Organ of Corti 1 (HEI-OC1) Cells. JARO: Journal of the Association for Research in Otolaryngology, 23(4), 467-489.

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Quantifying miRNA Expression by RT-qPCR

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TaqMan microRNA assays (Applied Biosystems) were used to quantify the relative expression level of hsa-miR-29a-3p (assay ID 002112), hsa-miR-128-3p (assay ID 002216), hsa-miR-223-3p (assay ID 000526) and rno-miR-3473 (assay ID. 475642). U6 snRNA (assay ID 001973) was used as an internal control. cDNA was synthesized using the Taqman miRNA RT Kit (Applied Biosystems). Total RNA (10 ng/mL) in 5 µL of nuclease-free water was added to 3 µL of 5× RT primer, 1.5 µL of 10× reverse transcriptase buffer, 0.15 µL of 100 mM dNTP, 0.19 µL of RNase inhibitor, 4.16 µL of nuclease-free water and 50 U of reverse transcriptase in a total volume of 15 µL. The reaction was performed in triplicate for 30 min at 16 °C, 30 min at 42 °C and 5 min at 85 °C. Analyses were performed using an Applied Biosystems 7500 Fast Real-Time PCR system and software (Thermo Fisher Scientific Inc., Waltham, MA, USA). The reaction mixture was prepared using the TaqMan Fast Universal PCR master mixture (Thermo Fisher Scientific Inc.). Thermal cycling conditions were as follows: 45 cycles of 95 °C for 15 s and 60 °C for 1 min.

Uemura R., Murakami Y., Hashimoto A., Sawada A., Otani K., Taira K., Hosomi S., Nagami Y., Tanaka F., Kamata N., Yamagami H., Tanigawa T., Watanabe T., Taguchi Y.H, & Fujiwara Y. (2017). Expression of Serum Exosomal and Esophageal MicroRNA in Rat Reflux Esophagitis. International Journal of Molecular Sciences, 18(8), 1611.

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miRNA-326 Expression Analysis by qRT-PCR

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Single-stranded cDNA was synthesized from purified RNA samples by the TaqMan miRNA RT Kit (Applied biosystems, California, USA) according to the manufacturer instructions using SimpliAmp thermal cycler (Applied biosystems, USA).
Relative expression levels of miRNA-326 and the endogenous reference (RNU6B) were carried out by QR-PCR using the ready-made assays (ThermoFisher scientific, USA, Cat No. 4427975) [17 (link)].
The QR-PCR mixture contained 10 µl TaqMan universal master mix, 1 µl primers and probes, 7 µl RNase-free water and 2 µl cDNA in a total reaction volume 20 µl.PCR was done under the following conditions: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min on Rotor Gene real time PCR system (Qiagen, USA).

Elgendy W., Swelem R., Aboudiba N, & Elwafa R.A. (2022). Role of MicroRNA-326 and its Target Genes Bcl-xL and Bak as Potential Markers in Platelet Storage Lesion in Blood Banks. Indian Journal of Hematology & Blood Transfusion, 38(4), 731-738.

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Quantitative Analysis of miRNA Expression

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Total RNA extracted from cells with the Qiagen miRNeasy Mini kit (Qiagen) was reversely transcribed to complementary DNA by using the TaqMan miRNA RT Kit and stem-loop RT primers (Applied Biosystems). MiRNA expression levels were tested using the TaqMan PCR kit as implemented in the ABI 7900 real-time PCR System (Applied Biosystems). To assess the mRNA expression levels of miR-155-5p target genes, SYBR PCR Master Mix reagent kits (TaKaRa) were used according to the manufacturer's instructions. The results of miRNA and mRNA expression were normalized using the threshold cycle (Ct) of U6 and β-actin, respectively. All reactions, including no-template controls, were performed in triplicate. Specific primers for amplification are shown in Supplementary Table S7.

Xie K., Ma H., Liang C., Wang C., Qin N., Shen W., Gu Y., Yan C., Zhang K., Dai N., Zhu M., Wu S., Wang H., Dai J., Jin G., Shen H, & Hu Z. (2015). A functional variant in miR-155 regulation region contributes to lung cancer risk and survival. Oncotarget, 6(40), 42781-42792.

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Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was isolated using the RNeasy Mini kit (QIAGEN), and cDNA was synthesized from equivalent total RNA using a SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen) according to the manufacturer's instructions. Primers used for amplification of target genes are listed in Supplementary Table 2. Amplification was carried out using an iCycler IQ Real-Time PCR Detection System, and cycle threshold (Ct) values were tabulated in duplicate for each gene of interest in each experiment. “No template” (water) controls were used to ensure minimal background contamination. Using mean Ct values tabulated for each gene, and paired Ct values for β-actin as a loading control, fold changes for experimental groups relative to assigned controls were calculated using automated iQ5 2.0 software (Bio-rad). For amplification of viral miRNAs, cDNA was synthesized using the Taqman miRNA RT kit (Applied Biosystems), and qPCR was performed using the Taqman MicroRNA Assays kit (Applied Biosystems) and a 7500 Real Time PCR System. Fold changes for microRNA were calculated using paired Ct values for RNU6B as recommended by the manufacturer (Applied Biosystems).

Cao Y., Qiao J., Lin Z., Zabaleta J., Dai L, & Qin Z. (2017). Up-regulation of tumor suppressor genes by exogenous dhC16-Cer contributes to its anti-cancer activity in primary effusion lymphoma. Oncotarget, 8(9), 15220-15229.

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Quantification of Exosomal miRNA Expression

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The expression of cellular exosomal miRNAs was assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was isolated from 400 μL exosome sample using the miVana PARISTM (Thermo Fisher Scientific, Waltham, WA, USA), according to the manufacturer’s instructions. cDNA synthesis was performed using the TaqMan miRNA RT kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed using TaqMan^® Universal PCR Master Mix II and monitored in real time using an RG-6000 real-time PCR. The cycle threshold (Ct) values were calculated using Rotor-Gene 6000 series software 1.7 (Corbett Research, Sydney, Australia). The relative expression levels of miRNAs in plasma samples were normalized with the 2^−ΔΔCT method using miR-191 as the internal control [21 (link)].

Kim S., Kim G.H., Park S.J., Kwon C.H., I H., Lee M.W, & Lee B.E. (2022). Exosomal MicroRNA Analyses in Esophageal Squamous Cell Carcinoma Cell Lines. Journal of Clinical Medicine, 11(15), 4426.

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Quantitative RT-PCR Analysis of miR-155-5p

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RT-qPCR for miR-155-5p was performed using a stem-loop RT primer and TaqMan miRNA RT kit (miRBase ID: hsa-miR-155-5p; catalog no. 4440886; Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer’s protocols. The primer and the thermo cycling condition are given in Table 3. The small nuclear RNA U6 (U6; assay ID: 001973) was used for normalization (Table 3). The expression level was calculated based on the threshold cycle value using the 2−^ΔΔCq method [43 (link)]. For the cells, the fold change of miR-155-5p expression was performed relative to DMSO-treated cells. For WBCs, the fold change was measured relative to age-matched controls.

Al-Moghrabi N., Al-Showimi M., Al-Yousef N, & AlOtai L. (2023). MicroRNA-155-5p, Reduced by Curcumin-Re-Expressed Hypermethylated BRCA1, Is a Molecular Biomarker for Cancer Risk in BRCA1-methylation Carriers. International Journal of Molecular Sciences, 24(10), 9021.

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Quantification of miR-500a Expression in Colorectal Cancer

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The expression levels of miR-500a in the 14 CRC tissue samples and FHC, SW620 and W1417 cells were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In detail, total RNA was extracted from the 14 specimens and three cell lines using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The expression levels of miR-500a were then detected by TaqMan miRNA RT-Real Time PCR, as previously described according to the manufacturer's protocol (16 (link)). Single-stranded cDNA was synthesized using the TaqMan miRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), and then amplified using TaqMan Universal PCR Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.), with miRNA-specific TaqMan Minor Groove Binder probes (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (17 (link)). The following primers were used: PCR miR-500a forward, 5′-ACACTCCAGCTGGGTAATCCTTGCTACCTGG-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′, and small nuclear RNA was used for normalization (18 (link)). The conditions were as follows: 40 cycles of three-step PCR (95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec) following an initial denaturation at 95°C for 10 min.

Li Y, & Chen Z. (2020). The oncogenic role of microRNA-500a in colorectal cancer. Oncology Letters, 19(3), 1799-1805.

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Quantification of miRNA-30e Expression

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Total RNA was extracted from cells or blood vessel tissue using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription (RT) was performed with the TaqMan miRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 37°C for 30 min and 84°C for 10 sec. qPCR was executed using the SYBR-Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) cycling profile: 95°C for 20 sec, 40 cycles of amplification (95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec) and melt curve analysis. The primer sequences for RT-qPCR: miR-30e 5′-GGG CAG TCT TTG CTA CTG TAA AC-3′ and 5′-GCC GCT GTA AAC ATC CGA CT-3′; U6 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′ and 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′. The relative expression levels of microRNA-30e were calculated using the 2^−ΔΔCq method (15 (link)).
Total RNA was hybridized using SurePrint G3 Mouse Whole Genome Microarray (G4471A-021828; Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA). Images were quantified using Agilent Feature Extraction Software (version A.10.7.3.1; Agilent Technologies, Inc.).

Cheng Y., Zhou M, & Zhou W. (2019). MicroRNA-30e regulates TGF-β-mediated NADPH oxidase 4-dependent oxidative stress by Snai1 in atherosclerosis. International Journal of Molecular Medicine, 43(4), 1806-1816.

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Quantitative PCR for miR-126 Expression

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Reverse transcription-quantitative PCR (RT-qPCR) for miR-126 was performed using a stem-loop RT primer and TaqMan miRNA RT kit (catalog no. 4427975; Applied Biosystems; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol (Table I) and using the thermocycling conditions stated below. The small nuclear RNA U6 (U6; assay ID: 001973) was used for normalization, and all primers are stated in Table I. The expression level was calculated based on the threshold cycle value using the 2^−ΔΔCq method (35 (link)). The fold-change of miR-126 expression in patients and carriers was performed relative to controls.

Al-Showimi M., Al-Yousef N., Alharbi W., Alkhezayem S., Almalik O., Alhusaini H., Alghamdi A, & Al-Moghrabi N. (2022). MicroRNA-126 expression in the peripheral white blood cells of patients with breast and ovarian cancer is a potential biomarker for the early prediction of cancer risk in the carriers of methylated BRCA1. Oncology Letters, 24(2), 276.

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