Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections.

Author(s): Serth J, Kuczyk MA, Paeslack U, Lichtinghagen R, Jonas U

Publication: Am J Pathol, 2000, Vol. 156, Page 1189-96

PubMed ID: 10751344 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of prior staining of frozen renal carcinoma and prostate hyperplasia sections on DNA yields as determined by PicoGreen and PCR amplification of p53 exon 6. The effect of measuring concentration by spectrophotometer instead of PicoGreen was investigated using frozen sections. The relationship between DNA yield, as determined by PicoGreen, and the number of acetone fixed-leukocytes and volume of microdissected frozen tissue were also examined. After sectioning, all specimens were fixed with acetone but few other details of specimen preparation are included.

   Summary of Findings:

   Prior histological staining of frozen tissue sections with Mayer's alum hematoxylin or Weigert's iron hematoxylin resulted in lower DNA yields when measured by fluorometry compared to specimens that had not been previously stained. In addition, eosine and kernechrot dye interfered with the PicoGreen signal. When sections of a renal cell carcinoma were stained with an unspecified hematoxylin prior to DNA extraction, DNA yields were decreased 14-fold as determined by PicoGreen and 21-fold as determined by PCR amplification of p53 exon 6. Linear relationships between the flourescent units (PicoGreen) and the DNA concentration from sections as determined by spectrophotometer, the number of leukocytes used for DNA extraction, and the microdissected specimen volume were observed.

   Biospecimens

   • Tissue - Kidney
   • Tissue - Prostate
   • Cell - White Cells

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Neoplastic - Benign
   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Spectrophotometry
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   PCR Specific	Type of tissue stain	Hematoxylin Unstained
   Biospecimen Acquisition	Biospecimen location	Kidney Prostate Leukocytes
   PCR Specific	Targeted nucleic acid	p53 exon 6
   Fluorometry Specific	Type of tissue stain	Mayer's alum hematoxylin Weigert's iron Hematoxylin Giemsa Eosine Kernechtrot Unstained Hematoxylin and eosine
   Fluorometry Specific	Technology platform	PCR Spectrophotometer

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