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Related Experiment Videos

3,3'-Diaminobenzidine staining interferes with PCR-based DNA analysis.

Christian Dölle1,2, Laurence A Bindoff1,2, Charalampos Tzoulis3,4

  • 1Department of Neurology, Haukeland University Hospital, Bergen, Norway.

Scientific Reports
|January 21, 2018
PubMed
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3,3'-Diaminobenzidine (DAB) staining significantly inhibits quantitative PCR (qPCR) analysis of mitochondrial DNA. This interference creates bias in mitochondrial DNA copy number and deletion level estimations, impacting molecular analyses of stained tissues.

Area of Science:

  • Histology
  • Molecular Biology
  • Biochemistry

Background:

  • 3,3 '-Diaminobenzidine (DAB) is a common chromogen used in histological staining.
  • Stained tissues are frequently utilized for downstream molecular analyses, including quantitative PCR (qPCR).

Purpose of the Study:

  • To investigate the impact of DAB staining on qPCR-based mitochondrial DNA quantification.
  • To determine if DAB interferes with PCR analysis in different cell types.

Main Methods:

  • Microdissection of muscle fibers and neurons from sequential tissue sections.
  • Comparison of qPCR results from DAB-stained and unstained samples.
  • Use of exogenous DNA templates to assess the mechanism of interference.

Main Results:

Related Experiment Videos

  • DAB staining significantly inhibits qPCR-based mitochondrial DNA quantification.
  • This inhibition introduces bias in estimating mitochondrial DNA copy number and deletion levels.
  • The effect was observed in both muscle fibers and human brain neurons, indicating a general interference with PCR.

Conclusions:

  • DAB staining is incompatible with PCR-based quantification methods.
  • Previously reported results using DAB staining for PCR analysis may require re-evaluation.
  • DAB interferes with the DNA template rather than inhibiting DNA polymerase activity.