Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections.
Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.
Serth
J
J
Department of Urology/Research Laboratory, Medical School Hannover, Hannover, Germany. serth.juergen@mh-hannover.de
Kuczyk
M A
MA
Paeslack
U
U
Lichtinghagen
R
R
Jonas
U
U
D004273:DNA, Neoplasm / Q000032:analysis*; D004210:Dissection; D005260:Female; D005404:Fixatives; D005456:Fluorescent Dyes; D005557:Formaldehyde; D005615:Freezing; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D007962:Leukocytes / Q000737:chemistry; D009930:Organic Chemicals; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000473:pathology
D004273:DNA, Neoplasm; D005404:Fixatives; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D005557:Formaldehyde