Direct and repeat uses of tissue sections as templates for liquid-phase polymerase chain reaction amplification: applications and implications.
The liquid-phase polymerase chain reaction (PCR) technique is the most commonly used method for the amplification of genetic materials, although it requires the lysis of cells for DNA or RNA extraction, making it impossible to visualize the distribution and subcellular localization of the biomolecules. This study intended to assess whether tissue sections may be directly and repeatedly used as templates for liquid-phase PCR amplifications. Consecutive paraffin sections of breast tissues were placed on gamma-methacryloxypropyltrimethoxysilane-coated microscopic cover glasses perforated with a diamond knife into strips. After hematoxylin and eosin or immuno-staining, the strip of interest was inserted into a PCR tube for amplifications, and the adjacent strip with the same tissue was subject to microdissection, DNA extraction, and PCR amplifications. To use the strip repeatedly, it was transferred into a new PCR tube and amplified with a new primer set, after an initial amplification for 5 to 7 cycles. Then, initially amplified samples were amplified to a total of 40 cycles. An equal volume of PCR products from the strip and DNA extract were loaded side by side for electrophoresis and detection. The strip and DNA extract from the same tissue yielded a very comparable quality and quantity of PCR products with the same primer sets. The strip, however, could be repeatedly used for PCR amplifications with substantially more primer sets. In addition, the strip could be used for immunohistochemical or other molecular assays after PCR amplifications. Further studies with the same protocol on strips containing chromosomal spreads generated similar results.
Man
Yan-Gao
YG
Department of Gynecology and Breast Pathology, Armed Forces Institute of Pathology and American Registry of Pathology, 6825 16th Street, NW, Washington 20306-6000, USA. man@afip.osd.mil
Zhang
Hong
H
Vang
Russell S
RS
Strauss
Brian L
BL
Zhang
Lin
L
Gao
Chunling
C
D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D006652:Histological Techniques; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D004247:DNA