High quality of DNA retrieved for Southern blot hybridization from microwave-fixed, paraffin-embedded liver tissues.
To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.
Hsu
H C
HC
Department of Pathology, National Taiwan University Medical College, Taipei, Republic of China.
Peng
S Y
SY
Shun
C T
CT
D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004279:DNA, Viral / Q000302:isolation & purification*; D005404:Fixatives; D006511:Hepatitis B Antigens / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D008872:Microwaves; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010232:Paraffin; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000235:genetics
D004279:DNA, Viral; D005404:Fixatives; D006511:Hepatitis B Antigens; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050806:presurface protein 1, hepatitis B surface antigen; D010232:Paraffin