Development and Assessment of a Quantitative Reverse Transcription-PCR Assay for Simultaneous Measurement of Four Amplicons

Background: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Vol. 49; no. 9; pp. 1467 - 1475
Main Authors: Grace, Marcy B, McLeland, Christopher B, Gagliardi, Steven J, Smith, Jeffrey M, Jackson, William E., III, Blakely, William F
Format: Journal Article
Language:English
Published: Washington, DC Am Assoc Clin Chem 01.09.2003
American Association for Clinical Chemistry
Oxford University Press
Subjects:
bcl-2-Associated X Protein
Biological and medical sciences
Biomarkers
Blood
DNA Repair
DNA, Complementary - chemistry
DNA-Binding Proteins - blood
DNA-Binding Proteins - genetics
Dosimetry
GADD45 Proteins
Gene expression
Humans
Intracellular Signaling Peptides and Proteins
Investigative techniques, diagnostic techniques (general aspects)
Irradiation
Laboratories
Male
Medical sciences
Miscellaneous. Technology
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Proteins - chemistry
Proteins - genetics
Proteins - metabolism
Proto-Oncogene Proteins - blood
Proto-Oncogene Proteins - genetics
Proto-Oncogene Proteins c-bcl-2
Radiation
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction - methods
RNA - chemistry
RNA, Ribosomal, 18S - blood
RNA, Ribosomal, 18S - chemistry
Superoxide Dismutase - blood
Superoxide Dismutase - genetics
Human
RNA
Blood
Dose activity relation
Gene
Reference system
Amplicon
DNA
Clinical biology
Irradiation
Genetics
Technique
Molecular biology
Reverse transcription polymerase chain reaction
Comparative study
Quantitative analysis
ISSN:0009-9147, 1530-8561
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Summary:Background: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. Methods: We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl2, and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. Results: The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log10 cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of −3.322. Conclusions: This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.
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ISSN:0009-9147
1530-8561
DOI:10.1373/49.9.1467