Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors
Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors
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DOI：
10.1016/s1525-1578(10)60010-4
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发表时间：
2005-02-01
影响因子：
4.1
通讯作者：
Waldman, F
中科院分区：
文献类型：
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作者：
DeVries, S;Nyante, S;Waldman, F
预后和预测性基因组标记的鉴定需要对患者进行长期的临床随访。从保存的福尔马林固定、石蜡包埋的材料中提取高质量的DNA对此类研究至关重要。特别重要的是针对小组织样本的全基因组扩增的稳健可重复方法。这对于高分辨率分析方法尤其如此，因为不同的基因组区域和序列可能会有差异地扩增。我们已经测试了许多用于基于阵列的比较基因组杂交（CGH）的DNA扩增的方案，其中整个基因组的相对拷贝数以1至2mb的分辨率测量。使用不同量的新鲜和石蜡提取的正常和乳腺肿瘤输入DNA测试了随机引物扩增和简并寡核苷酸引物扩增方法。我们发现，随机引物扩增明显优于基于阵列的CGH简并寡核苷酸引物扩增上级。最好的质量和再现性强烈依赖于使用定量聚合酶链反应为基础的方法输入DNA的量的准确测定。使用50 ng输入DNA获得了可再现的高质量结果，一些样品仅用5 ng输入DNA就获得了高质量结果。我们的结论是，随机引物扩增的DNA分离石蜡切片是一个强大的和可重复的方法，基于阵列的CGH分析档案肿瘤样本。
identification of prognostic and predictive genomic markers requires long-term clinical follow-up of patients. Extraction of high-quality DNA from archived formalin-fixed, paraffin-embedded material is essential for such studies. Of particular importance is a robust reproducible method of whole genome amplification for small tissue samples. This is especially true for high-resolution analytical approaches because different genomic regions and sequences may amplify differentially. We have tested a number of protocols for DNA amplification for array-based comparative genomic hybridization (CGH), in which relative copy number of the entire genome is measured at 1 to 2 mb resolution. Both random-primed amplification and degenerate oligonucleotide-primed amplification approaches were tested using varying amounts of fresh and paraffin-extracted normal and breast tumor input DNAs. We found that randomprimed amplification was clearly superior to degenerate oligonucleotide-primed amplification for array-based CGH. The best quality and reproducibility strongly depended on accurate determination of the amount of input DNA using a quantitative polymerase chain reaction-based method. Reproducible and high-quality results were attained using 50 ng of input DNA, and some samples yielded quality results with as little as 5 ng input DNA. We conclude that randomprimed amplification of DNA isolated from paraffin sections is a robust and reproducible approach for array-based CGH analysis of archival tumor samples.