Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections - PubMed (original) (raw)
Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections
J Serth et al. Am J Pathol. 2000 Apr.
Abstract
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.
Figures
Figure 1.
Sensitivity of the PicoGreen assay using medium scale-purified genomic DNA. The indicated amount of DNA was incubated in quadruplicate with the PicoGreen reagent in a total volume of 800 μl, and the fluorescence emission was measured at 532 nm (excitation wavelength 480 nm). To calculate the sensitivity parameters the slope of the regression line was determined (see Table 1 ▶ ). Note that some of the bars indicating the positive and negative SD fall within the data symbols.
Figure 2.
Sensitivity of the PicoGreen assay using a dilutional series of total DNA extracted from a known number of leukocytes. The amount of DNA corresponding to the indicated number of cells was incubated in quadruplicate with the PicoGreen reagent. For calculation of the sensitivity parameters, the slope of the indicated regression line was determined (see Table 1 ▶ ).
Figure 3.
Microdissections of a fresh frozen section of a renal cancer specimen (original magnification ×63). Note that hematoxylin staining of sections was carried out after the microdissection of cells. The numbers of microdissection (30 and 31) refer to the sensitivity analysis of microdissections shown in Figure 4 ▶ .
Figure 4.
Sensitivity analysis using DNA from microdissections of renal cell cancer (▵) and cervical cancer specimens (•). The amount of DNA extracted from each microdissection was assayed by PicoGreen fluorescence. Fluorescence signals were plotted against the volume of microdissected cells as calculated from the measured microdissected areas and the thickness of the section. The slope of the regression line together with the blank value variation was used for calculation of the sensitivity parameters (see Table 1 ▶ ). ▴, Microdissections 30 and 31, as shown in Figure 3 ▶ .
Figure 5.
A: Effect of formalin treatment on DNA extraction efficiency of complete serial cryosections from a snap-frozen rat kidney, each experiment carried out in triplicate. A1: Control, untreated cryosections; A2: 10-minute treatment of cryosections with 4% (w/v) unbuffered formalin; A3: extraction of sections of the corresponding contralateral formalin-fixed and paraffin-embedded rat kidney specimen. B: Control incubation of human placental DNA with formalin. B1: No formalin; B2: 4% (w/v) formalin. The error bars indicate the positive and negative standard deviations, respectively.
Figure 6.
Screening for effects of various histological stainings on the amount of DNA that can be extracted with a uniform Proteinase K digestion procedure and serial cuttings of a prostatic hyperplasia specimen. *Fluorescence signals that might be biased because of an interference with fluorescence detection (see Table 2 ▶ ).
Figure 7.
Effect of Delafield’s, Weigert’s iron, and Mayer’s hemalum stainings on the yield of DNA extraction from serial cuttings of a rat kidney in comparison to unstained reference sections; all stains have been carried out in quadruplicate. The bars indicate the positive and negative SD, respectively.
Figure 8.
Fluorescence quantitation of identical dilutional series of complete DNA extractions obtained from two serial sections, either hematoxylin stained (○) or unstained (▪). Numbers 1–15 refer to dilutional steps, each of a constant factor of 2.153 corresponding to a dilutional factor of one order of magnitude per four lanes.
Figure 9.
Nested PCR amplification of the dilutional series described in Figure 8 ▶ . PCR of dilutional series deriving from the hematoxylin-stained section (+) or unstained control section (−) were analyzed by PAGE after 25 (A), 27 (B), and 29 (C) cycles of the second-round PCR. Designation of lanes is the same as described in Figure 8 ▶ . L and N refer to the length markers and negative controls, respectively. Note that for reasons of comparability some overloading of the (−) samples was necessary.
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