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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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Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

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Comparison of different standards for real-time PCR-based absolute quantification.

S Dhanasekaran¹, T Mark Doherty, John Kenneth

¹Infectious Diseases Unit, St. John's Research Institute, Koramangala, Bangalore-560034, India.

Journal of Immunological Methods

|January 30, 2010

Summary

Reliable standards are crucial for quantitative real-time PCR (qPCR). Cloned target sequences demonstrated superior stability over PCR products, ensuring more accurate absolute copy number quantification in diagnostic and research applications.

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Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Quantitative real-time PCR (qPCR) enables absolute quantification of nucleic acid targets.
Reliable standards are critical for the accuracy and reproducibility of qPCR assays.
Commonly used standards include PCR products and cloned target sequences.

Purpose of the Study:

To compare the storage stability of four common qPCR standards: purified and unpurified PCR products, and circular and linear cloned plasmid sequences.
To evaluate the impact of standard type on qPCR assay performance, including copy number variance, PCR efficiency, and regression analysis.
To determine the optimal standard for absolute quantification in qPCR applications.

Main Methods:

Four types of standards (purified/unpurified PCR products, circular/linear plasmids) were stored at 4°C and -20°C.
Standard stability was assessed over 14 days by measuring copy number variance, PCR efficiency, and regression coefficient (R²).
Experiments were repeated six times for each standard type and storage condition.

Main Results:

All tested standards showed degradation over time, impacting PCR efficiency and accuracy.
Cloned target sequences (plasmids) exhibited significantly greater stability compared to PCR products.
Degradation of standards led to substantial variance in absolute quantification results.

Conclusions:

The choice of standard significantly influences the reliability of qPCR absolute quantification.
Cloned target sequences are more stable and recommended over PCR products for robust qPCR assays.
Routine testing of standard quality and stability is essential for accurate qPCR results in research and diagnostics.