Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer
| Autor: | Kanokwan Bishop, Hiromi Tanaka, Brittney-Shea Herbert, Paige N. Dahlgren, Shatovisha Dey |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Male
0301 basic medicine Genome instability Cancer Research Eff PCR efficiency law.invention DNA deoxyribonucleic acid PCR polymerase chain reaction law Neoplasms Multiplex Telomere Shortening Polymerase chain reaction Aged 80 and over Tm melting temperature Middle Aged Telomere lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens T telomere quantity Blotting Southern Real-time polymerase chain reaction MCF-7 Cells Female IDC invasive ductal carcinoma HT29 Cells Adult Original article SDS sodium dodecyl sulfate S scg quantity Biology lcsh:RC254-282 Young Adult scg single-copy genes 03 medical and health sciences Cell Line Tumor Multiplex polymerase chain reaction Humans TE Tris-EDTA DCIS ductal carcinoma in situ Aged Southern blot EDTA ethylenediaminetetraacetic acid UCSC University of California Santa Cruz ALT alternative lengthening of telomeres Reproducibility of Results CV coefficient of variation DNA HCT116 Cells mcs multiple-copy sequences Molecular biology M mcs quantity 030104 developmental biology Ct cycle threshold Primer (molecular biology) Multiplex Polymerase Chain Reaction DIG digoxigenin MMQPCR monochrome multiplex quantitative PCR HeLa Cells |
| Zdroj: | Neoplasia: An International Journal for Oncology Research, Vol 20, Iss 5, Pp 425-431 (2018) Neoplasia (New York, N.Y.) |
| ISSN: | 1476-5586 |
| DOI: | 10.1016/j.neo.2018.02.007 |
| Popis: | Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|