Li P X, Cheng L, Wen D R, Wissmann P B, Cheng J, Grody W W, Cochran A J
Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, California, USA.
Diagn Mol Pathol. 1997 Feb;6(1):26-33. doi: 10.1097/00019606-199702000-00005.
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.
为评估逆转录（RT）原位聚合酶链反应（PCR）和RT聚合酶链反应（PCR）原位杂交技术在研究人类肿瘤细胞中的特异性和适用性，我们使用酪氨酸酶特异性引物检测了5种黑色素瘤细胞系和5种非黑色素瘤细胞系中的酪氨酸酶信使核糖核酸（mRNA）。对每个操作步骤进行了优化并严格控制，并将原位技术与液相RT-PCR的结果进行了比较。所有黑色素瘤细胞系均显示出非黑色素瘤细胞系中未见的核周细胞质反应的特定模式。RT原位PCR和RT-PCR原位杂交技术的结果与液相RT-PCR的结果完全一致。核糖核酸酶消化消除了细胞质染色，逆转录酶步骤的省略也有同样效果。在黑色素瘤和非黑色素瘤细胞系中均可见细胞核染色，这显然是修复复制和错误引物或非特异性扩增导致DNA合成的结果。高浓度的脱氧核糖核酸酶和长时间孵育均不能完全消除这种效应。通过RT原位PCR和RT-PCR原位杂交证明细胞质mRNA可特异性识别黑素细胞系细胞。
