Demonstration of cytoplasmic tyrosinase mRNA in tissue-cultured cells by reverse transcription (RT) in situ polymerase chain reaction (PCR) and RT PCR in situ hybridization.
To evaluate the specificity and applicability to the study of human tumor cells of the reverse transcription (RT) in situ PCR and RT polymerase chain reaction (PCR) in situ hybridization techniques, we examined five melanoma cell lines and five nonmelanoma lines for tyrosinase mRNA using primers specific for tyrosinase. Each procedural step was optimized and minutely controlled, and results from the in situ techniques and solution-phase RT-PCR were compared. All melanoma lines showed a specific pattern of perinuclear cytoplasmic reaction not seen in nonmelanoma lines. There was exact agreement between the results from the RT in situ PCR and RT-PCR in situ hybridization techniques and those from solution-phase RT-PCR. Ribonuclease digestion abolished cytoplasmic staining, as did omission of the reverse transcriptase step. Nuclear staining was seen in melanoma and nonmelanoma lines, apparently as a result of DNA synthesis from repair-replication and mispriming or nonspecific amplification. Neither high concentrations of deoxyribonuclease nor long incubation periods abolished this effect completely. Demonstration of cytoplasmic mRNA by RT in situ PCR and RT-PCR in situ hybridization specifically identifies cells of melanocytic lineage.
Li
P X
PX
Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, California, USA.
Cheng
L
L
Wen
D R
DR
Wissmann
P B
PB
Cheng
J
J
Grody
W W
WW
Cochran
A J
AJ
D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics; D046508:Culture Techniques; D003593:Cytoplasm / Q000201:enzymology; D006801:Humans; D017403:In Situ Hybridization; D008545:Melanoma / Q000201:enzymology / Q000235:genetics*; D014442:Monophenol Monooxygenase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D014442:Monophenol Monooxygenase; D012194:RNA-Directed DNA Polymerase