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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 Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Published on: November 14, 2017

Internal validation of human mitochondrial DNA quantification using real-time PCR.

Marc L Sprouse¹, Nicole R Phillips, Mark F Kavlick

¹Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX, 76107.

Journal of Forensic Sciences

|April 9, 2014

Summary

Quantifying mitochondrial DNA (mtDNA) is crucial for sequencing success. A validated real-time PCR assay precisely measures mtDNA quantity, guiding optimal template amounts for forensic analysis and identifying potential PCR inhibition.

Keywords:

forensic science inhibition studies internal validation mitochondrial DNA quantitative PCR sequence analysis

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Area of Science:

Forensic Science
Molecular Biology
Genetics

Background:

Accurate quantification of mitochondrial DNA (mtDNA) template is essential for successful DNA sequencing.
Variability in mtDNA quantity can impact the quality and completeness of sequence data obtained.
Optimizing template input is critical for forensic applications, particularly with challenging samples like bone and teeth.

Purpose of the Study:

To validate a human mitochondrial DNA (mtDNA) real-time quantitative PCR (qPCR) assay for precise quantification.
To determine the optimal mtDNA template quantity for successful HV1 and HV2 sequencing analysis.
To assess the assay's utility in detecting PCR inhibition and guiding sample purification strategies.

Main Methods:

Validation of a human mtDNA real-time quantitative PCR (qPCR) assay.
Assessment of assay reproducibility, precision, and limit of detection.
Testing the assay on 35 nonprobative bone and teeth evidence samples to establish recommended template quantities.

Main Results:

The mtDNA qPCR assay demonstrated high reproducibility, precision, and a sensitive limit of detection (0.0001 pg/μL).
Approximately 20 pg of mtDNA template is recommended for standard HV1 and HV2 sequencing.
As little as 0.013 pg of mtDNA can yield a complete profile with enhanced amplification methods.
The assay effectively detected PCR inhibition, indicating samples needing re-purification.

Conclusions:

The validated mtDNA qPCR assay is an excellent tool for accurate mitochondrial DNA quantification.
This assay aids in selecting the optimal analytical approach for successful mtDNA sequencing, especially in forensic casework.
The assay's ability to detect PCR inhibition improves the reliability of forensic DNA analysis.