de Ruijter Tim C, de Hoon Joep P J, Slaats Jeroen, de Vries Bart, Janssen Marjolein J F W, van Wezel Tom, Aarts Maureen J B, van Engeland Manon, Tjan-Heijnen Vivianne C G, Van Neste Leander, Veeck Jürgen
1] Division of Medical Oncology, Maastricht University Medical Center, Maastricht, The Netherlands [2] GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
1] GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands [2] Department of Pathology, Maastricht University Medical Centre, Maastricht, The Netherlands.
Lab Invest. 2015 Jul;95(7):833-42. doi: 10.1038/labinvest.2015.53. Epub 2015 Apr 13.
Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffin-embedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n=9) and breast cancer (n=11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ=0.9927 (s.d. ±0.0015)). Matched FF/FFPEr correlation was also high (ρ=0.9590 (s.d. ±0.0184)) compared with matched FF/FFPE (ρ=0.8051 (s.d. ±0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ±0.66) was comparable to FF samples (99.98%, s.d. ±0.019) and substantially lower in FFPE samples (82.31%, s.d. ±18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for molecular pathological epidemiology research on archived samples with limited tissue amount.
目前，用于检测健康组织和病变组织中DNA甲基化的全基因组方法需要来自新鲜冷冻（FF）样本的高质量DNA。然而，注释完善的临床样本大多是福尔马林固定、石蜡包埋（FFPE）组织，其中的DNA质量较差。为克服这一限制，我们旨在评估一种DNA修复方案，用于全基因组Infinium HumanMethylation450 BeadChip检测（HM-450K）。使用HM-450K分别检测了66份来自正常结肠（n=9）和乳腺癌（n=11）的DNA样本。分析包括匹配的FF/FFPE样本和技术重复样本。FFPE DNA分别采用（FFPEr）或不采用DNA修复方案（Illumina）进行处理。最终，使用巢式甲基化特异性PCR（MSP）在另外24份FFPE组织中验证了差异甲基化基因。总之，FFPEr重复样本之间的β值相关性很高（ρ=0.9927（标准差±0.0015））。与匹配的FF/FFPE样本（ρ=0.8051（标准差±0.1028））相比，匹配的FF/FFPEr样本之间的相关性也很高（ρ=0.9590（标准差±0.0184））。FFPEr样本中的探针检测率（98.37%，标准差±0.66）与FF样本（99.98%，标准差±0.019）相当，而在FFPE样本中则显著较低（82.31%，标准差±18.65）。样本存档时间长达10年时，检测的稳健性并未降低。我们还证明，仅使用100 ng DNA输入时，检测的稳健性也不会降低。所选的5个差异甲基化基因中有4个可通过MSP验证。未能通过PCR验证的基因在引物结合位点的CpG β值变化很大。总之，通过使用FFPE DNA修复方案，HM-450K检测能够为源自FFPE组织的DNA提供稳健、准确和可重复的结果，与FF组织获得的结果相当。最重要的是，差异甲基化基因可以使用更敏感的技术（如巢式MSP）进行验证，从而为组织量有限的存档样本的分子病理流行病学研究提供一个表观基因组学平台。
