qRT-PCR was carried out in duplicate using Taqman miRNA assays (Applied Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems). The PCR mixture (10 μL) contained 5 μL Taqman Universal PCR Mastermix (Applied Biosystems), 3.83 μL nuclease-free DEPC-treated water, 0.5 μL Taqman miRNA assay (Applied Biosystems), and 0.67 μL reverse-transcribed products. The reaction conditions were 40 amplification cycles of 95°C for 15 s and 60°C for 1 min in a 96-well optical plate. Cycle threshold (Ct) values were calculated by using same threshold cut-off values for each assay to prevent plate-to-plate variations while analyzing the data with the 7500 software package v.2.0.6 (Applied Biosystems). The expression levels of miRNAs were normalized to spiked-in cel-miR-39, and were calculated utilizing the 2-ΔCt method [22 (link)]. ΔCt was calculated by subtracting the Ct values of cel-miR-39 from the Ct values of the miRNA of interest.
Taqman mirna assay
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Japan
TaqMan miRNA assays are a set of lab equipment designed for the quantitative detection and analysis of microRNA (miRNA) expression. These assays utilize the TaqMan technology, which enables highly sensitive and specific quantification of miRNA levels in a range of sample types. The core function of TaqMan miRNA assays is to provide researchers with a reliable tool for measuring the expression of miRNA molecules, which play important roles in various biological processes and disease states.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (162 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (100 mentions)
Trizol, by Thermo Fisher Scientific (70 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (53 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (50 mentions)
Mirneasy mini kit, by Qiagen (44 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (34 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (31 mentions)
Primescript rt reagent kit, by Takara Bio (28 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (24 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (19 mentions)
Sybr premix ex taq, by Takara Bio (16 mentions)
Nanodrop, by Thermo Fisher Scientific (14 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (13 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (13 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (12 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (12 mentions)
Mirna reverse transcription kit, by Thermo Fisher Scientific (12 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (12 mentions)
726 protocols using taqman mirna assay
1
Quantitative miRNA Expression Analysis
2
miRNA and Ras Gene Expression Analysis
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Total miRNA from HCC cells or snap-frozen HepG2 xenografts was isolated using the mirVANA™ PARIS™ RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA). RNA (10 ng) was reverse-transcribed with the miRNA Reverse Transcription Kit (Applied Biosystems) and let-7a specific primers (TaqMan miRNA assay, Applied Biosystems).
Total RNA was extracted from HCC cells or snap-frozen HepG2 xenografts using the IllustraRNA spin Mini RNA Isolation Kit (GE Healthcare UK Limited, Amersham Place, Little Chalfont, UK). cDNA was synthesized using SuperScript TM III First-Strand Synthesis SuperMix for quantitative real-time reverse transcription PCR (qRT-PCR; Invitrogen Corporation, Carlsbad, CA, USA) and primers specific for the 3 human ras genes (TaqMan miRNA assay, Applied Biosystems).
Quantitative PCR was performed using RNU6 or GAPDH as a housekeeping control with an ABI Prism 7500 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, USA) and the Perkin-Elmer Biosystems analysis software in a manner consistent with the manufacturer’s instructions. Relative expression was calculated using the 2-ΔΔCTmethod [23 (link)].
Total RNA was extracted from HCC cells or snap-frozen HepG2 xenografts using the IllustraRNA spin Mini RNA Isolation Kit (GE Healthcare UK Limited, Amersham Place, Little Chalfont, UK). cDNA was synthesized using SuperScript TM III First-Strand Synthesis SuperMix for quantitative real-time reverse transcription PCR (qRT-PCR; Invitrogen Corporation, Carlsbad, CA, USA) and primers specific for the 3 human ras genes (TaqMan miRNA assay, Applied Biosystems).
Quantitative PCR was performed using RNU6 or GAPDH as a housekeeping control with an ABI Prism 7500 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, USA) and the Perkin-Elmer Biosystems analysis software in a manner consistent with the manufacturer’s instructions. Relative expression was calculated using the 2-ΔΔCTmethod [23 (link)].
3
Profiling miRNA Expression in Hyperthyroid Mice
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For the miRNA expression analysis, 10 ng of total RNA was reverse transcribed using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, CA, USA) and RT primers provided with the miR-1 and miR-208a TaqMan miRNA Assay (Applied Biosystems, CA, USA) according to the manufacturer's instructions. The miRNA expression was detected from the cDNA product using a TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems, CA, USA) and the TaqMan miRNA Assay. Small nuclear RNA U6 (SNRNA1973, Applied Biosystems, CA, USA) was used for the normalization of data. Amplification and detection were performed using the Master Cycler Realplex system (Eppendorf, Germany). Relative quantifications were calculated according to the Pfaffl method [37] .
Microarray analyses were performed by LC Sciences (TX, USA) using 2 μg of total RNA from the wild-type (TRβ WT ) or homozygous (TRβ Δ337T ) group at the end of the hyperthyroidism protocol.
Microarray analyses were performed by LC Sciences (TX, USA) using 2 μg of total RNA from the wild-type (TRβ WT ) or homozygous (TRβ Δ337T ) group at the end of the hyperthyroidism protocol.
4
Validation of miRNA Expression in Vascular Calcification
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We then confirmed miRNA expression of selected miRNA that controlled multiple genes and/or controlled pathways which are known to be important in vascular calcification, by real time PCR as previously described [17 (link)] using TaqMan miRNA assays (Applied Biosystems, Foster City, CA). Forty ng of total RNA were used for reverse transcription to synthesize complementary DNA using TaqMan miRNA-specific primers and the Taq reverse transcription kit (Applied Biosystems, Foster City, CA). Target-specific PCR primers (miR-667, miR-702, miR-3562, miR-3568 and miR-3584) were obtained from Applied Biosystems. Real-time PCR amplifications were performed using TaqMan miRNA assays (TaqMan MGP probes, FAM dye-labeled) using Applied Biosystems 7500 Real-Time PCR systems (Applied Biosystems). The cycle number at which the amplification plot crosses the threshold was calculated (CT), and the ΔΔCT method was used to analyze the relative changes in gene expression and normalized by U6, a non-human ubiquitous miRNA.
5
Molecular Analysis of Muscle Atrophy in CKD
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Total RNA from normal and CKD EDL was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction. Total RNA was eluted from the column in RNase-free water and stored at –80°C. The gene and miRNA expression was determined by real time PCR using TaqMan miRNA assays (Applied Biosystems, Foster City, CA). Target-specific PCR primers (Pax-7, MyoD, Myostatin, Myogenin, Atrogin 1, MuRF-1, miR-29b, Activin 2b, SOD-1, and SOD-2) were obtained from Applied Biosystems. Real-time PCR amplifications were performed using TaqMan miRNA assays (TaqMan MGP probes, FAM dye-labeled) using Applied Biosystems ViiA 7 Real-Time PCR systems (Applied Biosystems). The cycle number at which the amplification plot crosses the threshold was calculated (CT), and the ΔΔCT method was used to analyze the relative changes in gene expression and normalized by β-actin or U6 (RNA and miRNA, respectively).
6
Validating Biomarker Candidates via TaqMan miRNA Assays
7
Quantifying PCBP1 mRNA and miRNAs
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RNA was isolated using Trizol (Life Technologies, Shanghai, China) as per manufacturer instructions. The expression level of PCBP1 mRNA and GAPDH mRNA were detected by TaqMan miRNA assays (Life Technologies, Shanghai, China). MiRNA from cells and tissues were extracted by the mirVana miRNA isolation kit (ThermoFisher Scientific, Shanghai, China) according to the manufacturer instructions. The expression levels of indicated miRNAs and RNU6B were detected by TaqMan miRNA assays (Life Technologies, Shanghai, China). The −ΔΔCt method was used to analyze the data in each case and normalization was done to GAPDH and RNU6B expression for mRNA and miRNA, respectively.
8
Quantitative Analysis of Hippocampal miRNA-21 Expression
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Hippocampal miRNA-21 was measured by RT-PCR: (Nolan et al. 2006 (link)) Total RNA Extraction: Separation of total RNA from serum samples was carried out using the Qiagen® miRNeasy Mini Kit (Qiagen, CA) according to the manufacturer's instructions (Leontariti et al. 2020 (link)).
Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (QRT-PCR): The quantification of miRNA-21 and U6 expression was performed using the Talkman mRNA assay using a two-step RT-PCR.
Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (QRT-PCR): The quantification of miRNA-21 and U6 expression was performed using the Talkman mRNA assay using a two-step RT-PCR.
Reverse transcription (RT) step: cDNA was reverse transcribed from purified RNA samples by using specific miRNA stem-loop primers from the TaqMan miRNA assays and reagents from the TaqMan® miRNA reverse transcription kit (Applied Biosystem, USA).
Quantitative real-time PCR (qPCR) step: PCR products were amplified from cDNA samples using the TaqMan miRNA assays together with the TaqMan® universal PCR master mix (Applied Biosystem, USA). Thermocycling was done using Applied BiosystemsStepOne™ real-time PCR System. The relative expression was calculated using the 2−∆∆CT method.
9
RNA Isolation and Marker Analysis
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Trizol was used for RNA isolation from tissue specimens and cells. STEAP1, SNAI1, MMP9, GAPDH, and ACTB expression were detected via TaqMan miRNA assay (Life Technologies), with data being and miRNA data.
10
Quantifying miR-126 Expression
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