Quantification of individual miRNAs was performed with the TaqMan Human MiRNA Assay (Applied Biosystems) according to the manufacturer's instructions, using a QuantStudio™ 12K flex real-time PCR system (Applied Biosystems). After qPCR, the Ct value of each miRNA was normalized to that of the spike-in control, cel-miR-238. The copy numbers of individual miRNAs were calculated from a standard curve plotted using 5 to 5 × 106 copies of synthetic cDNA oligonucleotides. The analytical detection limit was defined as a Ct value of 40; miRNAs with Ct > 40 were considered undetectable.
Taqman human mirna assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan human miRNA assays are a set of real-time PCR assays designed to detect and quantify mature human microRNA (miRNA) molecules. These assays utilize TaqMan technology and provide a sensitive and specific method for miRNA expression analysis.
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Lab products found in correlation
Taqman mirna reverse transcriptase kit, by Thermo Fisher Scientific (2 mentions)
7000 system sds software v1, by Thermo Fisher Scientific (2 mentions)
Quantstudio 12k flex real, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using taqman human mirna assay
1
Quantitative miRNA Profiling by qPCR
2
Profiling miRNA Expression in Osteosarcoma
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Briefly, total RNA was extracted from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Two microliters of cDNA obtained from each sample was amplified and quantified by real-time PCR of TaqMan Human MiRNA Assay (Applied Biosystems) according to the manufacturer’s protocol. MiRNAs expressions relative to U6 were evaluated using the comparative cycle threshold (CT) method.
Association between the clinicopathological parameters and expression of miRNAs in the osteosarcoma patients
| Clinicopathological features | No. of cases | Expression of miR-130b | Expression of miR-218 | P value of miR-130b | P value of miR-218 | ||
|---|---|---|---|---|---|---|---|
| Low = 11 | High = 19 | Low = 17 | High = 13 | ||||
| Gender | 0.741 | 0.712 | |||||
| Male | 17 | 7 | 10 | 11 | 6 | ||
| Female | 13 | 4 | 9 | 6 | 7 | ||
| Age | 0.517 | 0.483 | |||||
| <24 | 12 | 4 | 8 | 7 | 5 | ||
| ≥25 | 18 | 7 | 11 | 10 | 8 | ||
| Tumor diameter (cm) | 0.013 | 0.019 | |||||
| ≤6 | 16 | 9 | 7 | 6 | 10 | ||
| >6 | 14 | 2 | 12 | 11 | 3 | ||
| Histology | 0.51 | 0.503 | |||||
| Conventional osteosarcoma | 20 | 6 | 14 | 11 | 9 | ||
| Others | 10 | 5 | 5 | 6 | 4 | ||
| Clinical stage | 0.009 | 0.007 | |||||
| I | 12 | 8 | 4 | 4 | 8 | ||
| II | 10 | 3 | 7 | 6 | 4 | ||
| III | 8 | 0 | 8 | 7 | 1 | ||
| Distant metastasis | <0.001 | <0.001 | |||||
| No | 18 | 8 | 10 | 7 | 11 | ||
| Yes | 12 | 3 | 9 | 10 | 2 | ||
3
Quantitative miRNA Expression Analysis
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Approximately, 35 ng of total RNA was reverse-transcribed into a 10-uL volume with the TaqMan miRNA reverse-transcriptase kit (Applied Biosystems) according to the manufacturer’s recommendations. Then, 3 uL of the reverse-transcription reaction was used in each of the real-time PCR assays. Analyses of a subset of miRNAs (miR-21, miR-222 and miR-145) were carried out in triplicates by means of the TaqMan human miRNA assays (Applied Biosystems) using 7000 system SDS software v1.2.3 (Applied Biosystems).
4
Quantitative Analysis of miR-122
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Approximately 35 ng of total RNA was reverse-transcribed in a 10-uL volume using the TaqMan miRNA reverse-transcriptase kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Three microliters of the reverse-transcription reaction was used in each of the real-time PCR assays. Analyses of miR-122 were carried out in triplicates by means of the TaqMan human miRNA assays (Applied Biosystems) using SDS 7000 system software v1.2.3 (Applied Biosystems, USA).
5
Quantification of Mature Urinary miRNAs
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In urine specimens (Figure 1 ), abundance of selected mature miR-148a, -365, -375, and -429 was evaluated by TaqMan Human MiRNA Assays (assay numbers 000470, 001020, 000564, and 001024, respectively, Applied Biosystems, Thermo Fisher Scientific). RT was performed using the TaqMan MicroRNA Reverse Transcription Kit according to the manufacturer's protocol in 15 μl volume, each reaction containing specific stem-loop RT primer and 5 μl of total RNA from urine. In all, 1.33 μl of RT product were further amplified in triplicates in 20 μl qPCR reaction volume using TaqMan Universal PCR Master Mix, sequence specific primers, and TaqMan probes. Reactions were performed on ViiA 7 Real Time PCR System (Applied Biosystems, Thermo Fisher Scientific).
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