Detection of ptc in archival formalin-fixed, paraffin-embedded tissues. Comparison of radiolabeled DNA hybridization and direct incorporation of digoxigenin-11-dUTP into RT-PCR products.
Archival pathological specimens are a source of RNA and DNA for clinical surveillance or retrospective studies. We employed a modification of the acid guanidium thiocyanate-phenol-chloroform extraction method for the recovery of total RNA from formalin-fixed, paraffin-embedded neoplastic thyroid tissue. The extracted RNA was used for reverse transcription of ptc and subsequent amplification of the complementary DNA (cDNA) by the reverse transcription-polymerase chain reaction (RT-PCR). In lieu of 32P-labeled DNA for hybridization studies, we supplemented the nucleotide pool in the amplification reaction with a modified pyrimidine, digoxigenin-11-dUTP. Digoxigenin-11-dUTP was incorporated directly into the PCR product, eliminating the need for hybridization, posthybridization washes, and prolonged autoradiography. These products were resolved by electrophoresis on agarose gels, Southern blotted to nylon membranes, and rapidly detected by chemiluminescence. This nonradioisotopic method has expedited and reduced the cost for molecular investigations with archival pathological specimens by providing equal sensitivity to or greater sensitivity than that of DNA-labeled radionuclides without the associated biological hazards.
Martin
R K
RK
Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, Washington 98431-5000.
Archer
K T
KT
Tuttle
R M
RM
D002291:Carcinoma, Papillary / Q000235:genetics*; D017931:DNA Primers; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005557:Formaldehyde; D006801:Humans; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D016612:Paraffin Embedding; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011519:Proto-Oncogenes / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D013961:Thyroid Gland / Q000473:pathology; D013964:Thyroid Neoplasms / Q000235:genetics*; D016707:Tissue Fixation; D001613:beta 2-Microglobulin
D017931:DNA Primers; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D001613:beta 2-Microglobulin; C070202:digoxigenin-11-deoxyuridine triphosphate; D005557:Formaldehyde; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D004076:Digoxigenin