The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue.
Encouraged by recent reports of extraction of diagnostically useful DNA from formalin-fixed tissues, we devised a study to determine which fixatives are suitable for DNA hybridization studies. Seven lymph node specimens (6 lymphomas and one reactive hyperplasia) were studied. Specimens were divided into portions, each of which was processed separately. Processing protocols followed were: 1) snap freezing; 2) immersion in formalin, ethanol, glutaraldehyde, B5 and methanol acetic acid (MAA) Michel's transport medium or phosphate buffered saline for 2, 24, 48 and 120 hrs.; 3) paraffin embedding following fixation. DNA was obtained by proteinase K digestion followed by phenolchloroform extraction. DNA was cleaved with restriction endonucleases (Eco RI and Bam HI), separated by agarose gel electrophoresis, after which Southern blot hybridization with radio-labelled probes for J-heavy and T-beta genes was performed. Fixation with formalin, glutaraldehyde and B5 resulted in poor yields of DNA. MAA produced degradation of DNA and Michel's medium and PBS gave low quantities and purity of extracted DNA. Ethanol fixation consistently permitted extraction of large amounts of high molecular weight DNA suitable for hybridization studies and compared favourably with the fresh-frozen 'gold standard'. We conclude that ethanol may be the fixative of choice when transport or storage conditions limit the availability of fresh frozen tissue for DNA hybridization studies.
D000085:Acetates; D019342:Acetic Acid; D002725:Chloroform; D004247:DNA / Q000302:isolation & purification*; D004273:DNA, Neoplasm / Q000302:isolation & purification; D000431:Ethanol; D005404:Fixatives; D005557:Formaldehyde; D005629:Frozen Sections; D005976:Glutaral; D006801:Humans; D008221:Lymphoid Tissue / Q000032:analysis* / Q000473:pathology; D000432:Methanol; D009693:Nucleic Acid Hybridization; D013997:Time Factors
D000085:Acetates; D004273:DNA, Neoplasm; D005404:Fixatives; C046544:methacarn; D005557:Formaldehyde; D000431:Ethanol; D002725:Chloroform; D004247:DNA; D019342:Acetic Acid; D005976:Glutaral; D000432:Methanol