Ferguson Anthea Elizabeth, Cohn Richard Julian, Ashton Lesley Jayne
Molecular Epidemiology Group, Children's Cancer Institute Australia, Randwick, NSW, Australia.
Diagn Mol Pathol. 2012 Jun;21(2):105-13. doi: 10.1097/PDM.0b013e3182340a78.
Formalin-fixed paraffin-embedded tissue (FFPET) samples are a potential source of DNA for molecular epidemiological studies. However, the use of FFPET samples can be restricted by the yield and quality of DNA isolated. The aim of this study was to examine whether FFPET biopsies from pediatric central nervous system tumors were a feasible alternative to archival frozen tissue when characterizing common gene polymorphisms. DNA was isolated from 50 frozen pediatric central nervous system tumor biopsies and matched FFPET samples. Real-time polymerase chain reaction (PCR) was used to quantify DNA and characterize GSTT1, GSTM1, GSTP1, and MTHFR gene polymorphisms. The use of whole-genome amplification (WGA) to increase DNA yields was also investigated. The results showed that DNA isolated from FFPET samples was more fragmented and provided smaller yields than DNA isolated from frozen samples. Attempts to increase the DNA yield from FFPET using WGA were unsuccessful. DNA from FFPET samples was successfully genotyped for the GSTP1 Ile105Val and MTHFR 677 C>T polymorphisms in 98% of samples and was 100% concordant with the results from frozen tissue. However, DNA from FFPET performed poorly in real-time PCR assays for GSTM1 and GSTT1 deletion polymorphisms. Our investigations show that DNA extracted from FFPET is substantially fragmented and not readily amplified using WGA. In addition, careful validation of PCR assays should be carried out due to the variable amplification of fragmented FFPET DNA.
福尔马林固定石蜡包埋组织（FFPET）样本是分子流行病学研究中DNA的潜在来源。然而，FFPET样本的使用可能会受到所分离DNA的产量和质量的限制。本研究的目的是检验在对常见基因多态性进行特征分析时，来自小儿中枢神经系统肿瘤的FFPET活检样本是否是存档冷冻组织的可行替代物。从50份冷冻的小儿中枢神经系统肿瘤活检样本及与之匹配的FFPET样本中分离DNA。使用实时聚合酶链反应（PCR）对DNA进行定量，并对GSTT1、GSTM1、GSTP1和MTHFR基因多态性进行特征分析。还研究了使用全基因组扩增（WGA）来提高DNA产量。结果表明，从FFPET样本中分离的DNA比从冷冻样本中分离的DNA片段化程度更高，产量更低。尝试使用WGA提高FFPET样本的DNA产量未成功。在98%的样本中，FFPET样本的DNA成功进行了GSTP1 Ile105Val和MTHFR 677 C>T多态性的基因分型，并且与冷冻组织的结果100%一致。然而，FFPET样本的DNA在针对GSTM1和GSTT1缺失多态性的实时PCR检测中表现不佳。我们的研究表明，从FFPET中提取的DNA片段化程度很高，并且使用WGA不易扩增。此外，由于FFPET片段化DNA的扩增情况可变，应仔细进行PCR检测的验证。
