3,3'-Diaminobenzidine (DAB) is a widely used chromogen in histological staining methods and stained tissue is often used in downstream molecular analyses such as quantitative PCR (qPCR). Using microdissected muscle fibers from sequential muscle sections stained by DAB-dependent and -independent methods, we show that DAB exerts a strong inhibitory effect on qPCR-based mitochondrial DNA quantification. This effect introduces a significant bias in the estimation of mitochondrial DNA copy number and deletion levels between DAB-positive and -negative fibers. We reproduce our findings in microdissected neurons from human brain tissue, suggesting a general effect of DAB staining on PCR analyses independent of the underlying tissue or cell type. Using an exogenous DNA template added to tissue samples we provide evidence that DAB-staining predominantly interferes with the tissue-derived DNA template rather than inhibiting DNA polymerase activity. Our results suggest that DAB-based staining is incompatible with PCR-based quantification methods and some of the previously reported results employing this approach should be reconsidered.

中文翻译：

---

3,3'-二氨基联苯胺染色会干扰基于PCR的DNA分析。

3,3'-二氨基联苯胺（DAB）是组织学染色方法中广泛使用的色原，被染色的组织通常用于下游分子分析，例如定量PCR（qPCR）。使用由DAB依赖性和非依赖性方法染色的连续肌肉切片中的显微解剖肌纤维，我们显示DAB对基于qPCR的线粒体DNA定量具有强大的抑制作用。这种效应在估计DAB阳性和阴性纤维之间的线粒体DNA拷贝数和缺失水平方面引入了显着的偏差。我们在人类脑组织的显微解剖神经元中重现了我们的发现，表明DAB染色对PCR分析的一般影响不依赖于基础组织或细胞类型。使用添加到组织样本中的外源DNA模板，我们提供证据表明DAB染色主要干扰组织衍生的DNA模板，而不是抑制DNA聚合酶活性。我们的结果表明，基于DAB的染色与基于PCR的定量方法不兼容，应重新考虑采用该方法的一些先前报道的结果。