Total RNA was isolated by using TRIzol or TRIzol LS reagent (Invitrogen) according to the manufacturer’s protocol. miRNA assays by real-time PCR were performed using specific TaqMan primers (Invitrogen). U6 small nuclear RNA (snRNA) was used as an endogenous control. Real-time analyses by two-step RT-PCR were performed for quantification of miRNA levels on a Bio-Rad CFX96 real-time system using an Applied Biosystems TaqMan chemistry-based miRNA assay system. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan universal PCR master mix, no AmpErase (Applied Biosystems) and the respective TaqMan reagents for target miRNAs. Samples were analyzed in triplicates. The comparative Ct method, which included normalization by the U6 snRNA, was used for relative quantification. For quantification of mRNA levels, 200 ng of total cellular RNA was subjected to cDNA preparation followed by qPCR by the SYBR Green method. Each sample was analyzed in triplicates using the comparative Ct method. mRNA levels were normalized with GAPDH as the loading control.
Taqman chemistry based mirna assay system
Manufactured by Thermo Fisher Scientific
The TaqMan chemistry-based miRNA assay system is a tool used for the detection and quantification of microRNA (miRNA) molecules. It utilizes TaqMan probe-based technology to enable sensitive and specific measurement of miRNA expression levels in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Trizol ls reagent, by Thermo Fisher Scientific (3 mentions)
Taqman universal pcr master mix no amperase, by Thermo Fisher Scientific (3 mentions)
Taqman primer, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Thermo Fisher Scientific (1 mentions)
5 protocols using taqman chemistry based mirna assay system
1
Quantification of miRNA and mRNA Levels
2
Quantitative Real-Time PCR of miRNAs
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Total RNA is isolated by using TriZol or TriZol LS reagent (Invitrogen) according to the manufacturer's protocol. MiRNA assays by real time PCR was performed using specific primers for human let-7a (assay ID 000377), human miR-122 (assay ID 000445), human miR-122* (assay ID 002130), human miR-16 (assay ID 000391). U6 snRNA (assay ID 001973) was used as an endogenous control. Real time analyses by two-step RT-PCR was performed for quantification of miRNA levels on Bio-Rad CFX96TM real time system using Applied Biosystems Taqman chemistry-based miRNA assay system. Cycles were set according to manufacter's protocol. Samples were analyzed in triplicates. The comparative Ct method which included normalization by the U6 snRNA was used for relative quantification.
3
Quantification of miRNA and mRNA Levels
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Total RNA was isolated by using TriZol or TriZol LS reagent (Invitrogen) according to the manufacturer's protocol. MiRNA assays by real time PCR was performed using specific Taqman primers (Invitrogen). U6 snRNA was used as an endogenous control. Real time analyses by two-step RT-PCR was performed for quantification of miRNA levels on Bio-Rad CFX96TM real time system using Applied Biosystems Taqman chemistry based miRNA assay system. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan® Universal PCR Master Mix No AmpErase (Applied Biosystems) and the respective TaqMan® reagents for target miRNA. Samples were analyzed in triplicates. The comparative Ct method which included normalization by the U6 snRNA was used for relative quantification. For quantification of mRNA level, 200ng of total cellular RNA was subjected to cDNA preparation followed by qPCR by SYBR Green method. Each sample was analyzed in triplicates using comparative Ct method. Each mRNA levels were normalized with GAPDH as loading control.
4
Quantification of miRNA by Two-Step RT-PCR
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Real‐time analyses by two‐step RT–PCR were performed for quantification of miRNA using Thermo Scientific TaqMan chemistry‐based miRNA assay system. It was performed with 25 ng of cellular RNA using specific primers for human let‐7a (assay ID 000377). U6 snRNA (assay ID 001973) was used as an endogenous control. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan® Universal PCR Master Mix No AmpErase (Thermo Scientific) and the respective TaqMan® reagents for target miRNA. Samples were analyzed in PCR triplicates from two biological replicates. The comparative Ct method which included normalization, by the U6 snRNA, was used for each cell type for plotting of mean values with SD.
5
Quantifying miRNA Expression by RT-PCR
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Real-time analyses by two-step RT–PCR were carried out to quantify miRNA expression using the Thermo Scientific TaqMan chemistry-based miRNA assay system as performed earlier [56 (link)]. 25 ng of cellular RNA were used along with specific primers for human miR-122 (assay ID 000445). U6 snRNA (assay ID 001973) was used as an endogenous Control. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan® Universal PCR Master Mix No AmpErase (Thermo Scientific) and the respective TaqMan® reagents for target miRNA. Samples were analyzed in PCR triplicates from at least two biological replicates. The comparative Ct method which included normalization by the U6 snRNA, was used for each cell type for plotting of mean values with s.d.
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