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Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues
M P Burton1, B G Schneider, R Brown
1Wilford Hall Medical Center, University of Texas Health Science Center, San Antonio, USA.
Abstract:
We evaluated the effect of six different histologic stains on the productivity of PCR amplification of DNA isolated from paraffin-embedded tissue samples. The tissue was collected from glass slides by microdissection techniques, whereby tiny portions of tissue are visually identified through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amplification depended on the type of histologic stain that was used to facilitate microscopic visualization of the undeparaffinized tissue section. The best results were obtained with methyl green and nuclear fast red, while Wright's stain yielded less PCR product. Two other stains, Evans blue and light-green SF yellowish (also known as the counterstain for geomori methenamine silver stain) yielded sufficient PCR products; however, their staining characteristics did not afford satisfactory visualization of nuclear chromatin to discriminate between benign and malignant cells. Our most significant finding was that a commonly used histologic stain, hematoxylin, failed to produce DNA templates that could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting material for PCR. Among the remaining five stains that were evaluated, the best choice depends on the differential staining characteristics of the cells to be dissected.
Insights
Histologic stains impact DNA amplification from tissue. Hematoxylin stains hinder PCR, while methyl green and nuclear fast red stains improve it for microdissection analysis.
Area of Science:
- Molecular Biology
- Histology
- Genetics
Background:
- Paraffin-embedded tissues are crucial for molecular analysis.
- Histologic stains aid in visualizing tissue structures for microdissection.
- The compatibility of stains with downstream DNA extraction and PCR amplification is critical.
Purpose of the Study:
- To evaluate the impact of six common histologic stains on PCR amplification success.
- To identify optimal stains for DNA extraction and PCR from microdissected tissues.
- To determine if specific stains inhibit or enhance DNA amplification.
Main Methods:
- DNA was isolated from paraffin-embedded tissue sections stained with six different histologic stains.
- Microdissection techniques were used to collect specific tissue portions.
- PCR amplification was performed on extracted DNA to assess productivity.
Main Results:
- PCR amplification success varied significantly based on the histologic stain used.
- Methyl green and nuclear fast red stains yielded the best PCR results.
- Hematoxylin stains failed to produce consistently amplifiable DNA templates.
- Wright's stain produced less PCR product, while Evans blue and light-green SF yellowish showed moderate success but poor cellular visualization.
Conclusions:
- Hematoxylin stains should be avoided for preparing tissues for PCR amplification.
- Methyl green and nuclear fast red are recommended for optimal PCR success from microdissected tissues.
- Stain selection should consider both DNA amplification efficiency and cellular visualization for accurate microdissection.