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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Nanoliter high-throughput RT-qPCR: a statistical analysis and assessment.

James M Dixon¹, Mariusz Lubomirski, Dhammika Amaratunga

¹Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Welsh and McKean Roads, Spring House, PA 19477, USA.

|June 2, 2009

Summary

A new nanoliter fluidic system enables high-throughput quantitative PCR (qPCR) assays for biomarker discovery. This scalable technology enhances drug development by preserving RT-qPCR precision for large gene panels and sample cohorts.

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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Gene expression biomarkers are crucial for drug development and diagnostics.
Quantitative reverse transcriptase PCR (RT-qPCR) is a validated method for gene expression analysis.
Current RT-qPCR methods face scalability challenges with increasing gene numbers and sample sizes.

Purpose of the Study:

To evaluate a novel nanoliter fluidic system for high-throughput RT-qPCR.
To assess the system's performance in terms of accuracy, precision, sensitivity, and reproducibility.
To determine the system's potential for advancing pharmaceutical research.

Main Methods:

A nanoliter fluidic system capable of 3072 simultaneous RT-qPCR assays was utilized.
Gene expression was measured in two adult human tissue types.
Assays assessed reproducibility, accuracy, precision, specificity, sensitivity, false positive rate (FPR), and false negative rate (FNR).
Kinase gene expression differences were analyzed to reflect tissue and dosage variations.

Main Results:

The nanoliter fluidic system demonstrated high performance across key analytical metrics.
Reproducibility, accuracy, precision, specificity, and sensitivity were confirmed.
Low false positive and false negative rates were observed for expressed transcripts.
Significant differences in kinase gene expression were detected between tissues and dosages.

Conclusions:

The evaluated nanoliter fluidic system is a scalable and precise tool for high-throughput RT-qPCR.
This technology holds significant potential for biomarker discovery in pharmaceutical research and development.
The system effectively addresses the limitations of traditional RT-qPCR for large-scale gene expression studies.