Detection of Copy Number Changes in DNA from Formalin Fixed Paraffin Embedded Tissues Using Paralogue Ratio Tests
| Autor: | Linda Potter, Abdlrzag Ehdode, Gerald Saldanha, Lovesh Dyall, J.H. Pringle, Nisreen Hathiari, Danielle Bury, Edward J. Hollox |
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| Rok vydání: | 2011 |
| Předmět: |
Male
Tissue Fixation Adolescent DNA Copy Number Variations Somatic cell Molecular Sequence Data Genomics Polymerase Chain Reaction Analytical Chemistry law.invention Young Adult chemistry.chemical_compound Pregnancy law Cell Line Tumor Formaldehyde Sequence Homology Nucleic Acid Genotype Chromosomes Human Humans Microdissection Polymerase chain reaction Paraffin Embedding Base Sequence Chromosome DNA Middle Aged Reference Standards Molecular biology chemistry Genetic Loci Nucleic acid Female |
| Zdroj: | Analytical Chemistry. 83:3484-3492 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | The purpose of this study was to evaluate whether paralogue ratio tests (PRT) using real-time PCR can accurately determine the DNA copy number (CN) using formalin fixed paraffin embedded (FFPE) tissue. Histopathology diagnostic archives are an enormous resource of FFPE tissue, but extracted DNA is of poor quality and may be unsuitable for CN assessment, thus representing a missed opportunity for studies of genetic association and somatic change in cancer in large cohorts of easily accessible samples. Assays with paralogues on chromosomes 18 and 20 (18|20 PRT) and chromosomes 13 and X (13|X PRT) were tested using archived FFPE pathology samples with known CN, including tonsil, placentae, and FFPE melanoma cell lines. The assay proved accurate over the dynamic range from 1:1 to 1:3 and gave precise results when repeated four times over several weeks. The precision of the assay was marginally reduced once the CT value for 10 ng of FFPE DNA increased above 30 cycles, reflecting importance of DNA quality. The assays distinguished changes in CN ratio with high sensitivity and specificity. The 13|X PRT could detect cells with distinct genotypes microdissected from within the same FFPE sample. Therefore, PRTs are suitable for analyzing CN in FFPE tissues. |
| Databáze: | OpenAIRE |
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