University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
PLoS One. 2013 Oct 14;8(10):e77771. doi: 10.1371/journal.pone.0077771. eCollection 2013.
Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.
福尔马林固定和石蜡包埋（FFPE）组织是生物标志物研究和临床常规诊断的宝贵资源。然而，由于固定过程，它们的核酸会降解。由于遗传和表观遗传研究通常需要 PCR 扩增，这种降解会显著阻碍其使用，降低 PCR 的稳健性或需要短的扩增子。在常规实验室医学中，高度稳健的 PCR 性能对于遗传和表观遗传生物标志物的临床应用是强制性的。因此，使用 FFPE 组织中的 DNA 来提高 PCR 性能的方法是非常需要的，并且更广泛地受到关注。通过使用不同大小的 PCR 片段进行 qPCR 和常规 PCR，研究了源自 FFPE 组织的模板 DNA 对 PCR 性能的影响。通过琼脂糖凝胶电泳分析 DNA 片段化。这项研究表明，PCR 扩增不良部分是由于 FFPE 组织的碎片化 DNA 抑制了 DNA 聚合酶，而不仅仅是由于缺乏足够完整性的完整模板分子。通过增加聚合酶浓度、dNTP 浓度和 PCR 延伸时间，成功地最小化了这种 PCR 抑制，从而允许对更大的扩增子进行稳健的扩增。这在基因组模板 DNA 以及用于 DNA 甲基化分析的亚硫酸氢盐转化模板 DNA 中均得到了证明。总之，使用 FFPE 组织中的 DNA 进行 PCR 会受到抑制，通过调整 PCR 条件可以缓解这种抑制，从而显著提高 PCR 性能，减少变异性并生成更大的扩增子。本研究提出的克服这种 PCR 抑制的解决方案对于临床化学和实验室医学具有巨大的价值。
