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 clinic al surveillance or retrospective studies. We employed a modification o f the acid guanidium thiocyanate-phenol-chloroform extraction method f or the recovery of total RNA from formalin-fixed, paraffin-embedded ne oplastic thyroid tissue. The extracted RNA was used for reverse transc ription of ptc and subsequent amplification of the complementary DNA ( cDNA) by the reverse transcription-polymerase chain reaction (RT-PCR). In lieu of P-32-labeled DNA for hybridization studies, we supplemente d the nucleotide pool in the amplification reaction with a modified py rimidine, digoxigenin-11-dUTP. Digoxigenin-11-dUTP was incorporated di rectly into the PCR product, eliminating the need for hybridization, p osthybridization washes, and prolonged autoradiography. These products were resolved by electrophoresis on agarose gels, Southern blotted to nylon membranes, and rapidly detected by chemiluminescence. This nonr adioisotopic method has expedited and reduced the cost for molecular i nvestigations with archival pathological specimens by providing equal sensitivity to or greater sensitivity than that of DNA-labeled radionu clides without the associated biological hazards.