Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer.
Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer.
复制标题
DOI：
10.1016/j.neo.2018.02.007
复制
发表时间：
2018-05
期刊：
影响因子：
--
通讯作者：
Tanaka H
中科院分区：
文献类型：
--
作者：
Dahlgren PN;Bishop K;Dey S;Herbert BS;Tanaka H
在大多数癌细胞中已经观察到过度的端粒缩短。端粒定量聚合酶链反应（qPCR）检测已成为流行病学研究的重要工具，检查衰老，压力和其他因素对端粒长度的影响。目前的端粒qPCR方法通过扩增端粒序列产物并用单拷贝基因产物标准化来分析端粒的相对长度。然而，目前的端粒qPCR并不总是反映癌症DNA中的绝对端粒长度。由于癌细胞中基因组的不稳定性，我们假设使用单拷贝基因（scg）对癌症DNA中的数据进行归一化不太准确，并且需要新的引物组来更好地代表癌症DNA中的相对端粒长度。我们首先证实了癌细胞在不同的scg中具有不同的拷贝率，这意味着DNA是非整倍体的。通过使用在整个基因组中扩增多拷贝序列（mcs）的新引物组，端粒qPCR结果表明，mcs引物与作为正常DNA中的参考引物的scg引物是可互换的。通过比较来自传统的Southern印迹方法的结果（作为酶）和来自使用mcs引物的单色多重qPCR的结果（作为T/M比率），我们验证了T/M比率与来自Southern印迹分析的绝对端粒长度高度相关。总之，mcs引物能够准确地代表癌症DNA样本中的端粒长度。这些结果将允许分析癌DNA中的端粒，并开发新的、侵入性较小的癌症诊断工具。
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.
登录
查看更多内容
影响因子：
14.9
作者：
Cawthon RM
通讯作者：
Cawthon RM
影响因子：
16.6
作者：
Lai TP;Zhang N;Noh J;Mender I;Tedone E;Huang E;Wright WE;Danuser G;Shay JW
通讯作者：
Shay JW
DOI：
10.1016/j.mrfmmm.2011.04.003
发表时间：
2012-02-01
影响因子：
2.3
作者：
Aubert, Geraldine;Hills, Mark;Lansdorp, Peter M.
通讯作者：
Lansdorp, Peter M.
影响因子：
14.9
作者：
Lee M;Hills M;Conomos D;Stutz MD;Dagg RA;Lau LM;Reddel RR;Pickett HA
通讯作者：
Pickett HA
影响因子：
6.6
作者：
Hattinger, CM;Reverter-Branchat, G;Serra, M
通讯作者：
Serra, M