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Objective: Most studies of the reverse transcriptase in situ polymerase chain reaction (PCR) technique have reported rsults from assessments of cultured cells, frogen sections, and cytospin preparations. For application to routine diagnosis, it will be necessary to adapt the technique for use formalin fixed, paraffin embedded tissues, the materials that are generally available. We evaluated the feasibility of such an approach. Methods: We applied RT in situ PCR archival formalin fixed, paraffin embedded tissue from human cutaneous melanoma using a primer for tyrosinase mRNA, by means of surgical pathology archival material from 25 UCLA patients: 15 tissues form primary and metastetic melanoma, 7 from nonmelanocytic tumors, including cancer of the lung, colon, kidney and skin and a thyroid adenoma, and 3 nontumarous tissues. The materials and methods in details included: (1)Cell button preparation ;(2) Archival melanoma tissues; the original slides from all tissues were reviewed and the diagnosis confirmed,(3) Tissue section preparation ;(4) In situ RT, (5) In situ PCR,(6) Immunodetection of PCR products. Results: Seven of 15 melanoma tissues gave a strong positive signal, 5 gave a weak signal, and 3 were negative. None of the 10 nonmelanoma tissue gave a positive signal. The specific reaction product was mainly located in the cyfoplasm. None of the nonmelanocytic tumors or normal tissues demonstrated this pattern of cytoplasmic staining. Some nonspecific nuclear staining was observed in melanocytic and nonmelanocytic tumors and must not be overread as a true positive result. Conclusion: By the apprication of RT in situ PCR, we have demonsted expression of tyrosinase mRNA in archival fomalin fixed, paraffin embedded tissue sections of human cutaneous melanoma and comfirmed the presence of tyrosinase mRNA in melanoma cell lines. All nonmelanoma tissues and nonmelanoma cell lines were negative. In this study our results confirm the usefulness that the presece of tyrosinate mRNA was specific to cells of melanocytic lineage. Although our data indicate that RT in situ PCR can be applied to archival tissue sections, the negativity of three melanomas and the weak positivity of five melanomas suggests that further technical improvement will be necessory to allow widespread routine use of this technique for diagnostic purposes. The authors conclude that it is possibe to detect tyrosinase mRNA in formalin fixed, paraffin embedded tissue sections of melanoma, but the technique remains to demanding for routine application.
Objective: Most studies of the reverse transcriptase in situ polymerase chain reaction (PCR) technique have reported rsults from assessments of cultured cells, frogen sections, and cytospin preparations. For application to routine diagnosis, it will be necessary to adapt the technique for use formalin Methods: We applied the feasibility of such an approach for such an approach. We applied RT in situ PCR archival formalin fixed, paraffin embedded tissue from human cutaneous melanoma using a primer for tyrosinase mRNA, by means of surgical pathology archival material from 25 UCLA patients: 15 tissues form primary and mettictic melanoma, 7 from nonmelanocytic tumors, including cancer of the lung, colon, kidney and skin and a thyroid adenoma, and 3 nontumarous tissues. The materials and methods in details included: (1) Cell button preparation; (2) Archival melanoma tissues; the original slides from all tissues were reviewed (4) In situ RT, (5) In situ PCR, (6) Immunodetection of PCR products. Results: Seven of 15 melanoma tissues gave a strong positive signal, 5 gave a None of the 10 nonmelanoma tissue gave a positive signal. The specific reaction product was mainly located in the cyfoplasm. None of the nonmelanocytic tumors or normal tissues demonstrated this pattern of cytoplasmic staining. Some nonspecific nuclear staining was observed in melanocytic and nonmelanocytic tumors and must not be overread as a true positive result. Conclusion: By the apprication of RT in situ PCR, we have demonsted expression of tyrosinase mRNA in archival fomalin fixed, paraffin embedded tissue sections of human cutaneous melanoma and comfirmed the presence of tyrosinase mRNA in melanoma cell lines. All nonmelanoma tissues and nonmelanoma cell lines were negative. In this study our results confirm the usefulness that the preWhile our data indicate that RT in situ PCR can be applied to archival tissue sections, the negativity of three melanomas and the weak positivity of five melanomas suggests that further technical improvement will be necessory to allow widespread routine use of this technique for diagnostic purposes. The authors conclude that it is possibe to detect tyrosinase mRNA in formalin fixed, paraffin embedded tissue sections of melanoma, but the technique remains to be demanding for routine application.