Cairns M T, Church S, Johnston P G, Phenix K V, Marley J J
Department of Oncology, Queen's University of Belfast, Belfast City Hospital Tower, UK.
Oral Dis. 1997 Sep;3(3):157-61. doi: 10.1111/j.1601-0825.1997.tb00028.x.
To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level.
We describe the isolation of RNA from 8 microns sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RI alpha gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cells.
The S14 gene, the TK gene and the RI alpha gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species.
RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.
评估利用存档口腔黏膜组织在核糖核酸（RNA）水平检测基因表达的可行性。
我们描述了从福尔马林固定石蜡包埋的口腔黏膜组织8微米切片中分离RNA的方法。RNA经逆转录，通过聚合酶链反应（PCR）扩增三个候选基因。核糖体蛋白S14基因是一种管家基因，已在多种定量PCR方案中用作内标。胸苷激酶（TK）基因在大多数组织中低水平表达，且基因组结构有充分记录，是区分基因组DNA和cDNA的有用工具。据报道，RIα基因在许多癌细胞系、恶性组织和体外转化细胞中过表达。
成功从福尔马林固定石蜡包埋的组织切片中扩增出cAMP依赖性蛋白激酶（PKA）的S14基因、TK基因和RIα基因。TK引物对是明确鉴定RNA衍生物种的有用辅助工具。
从存档口腔黏膜组织中提取了适合逆转录（RT）-PCR的RNA。这应能对该材料中转录的肿瘤抑制基因和癌基因进行快速序列分析。此外，所述的RT-PCR方法可能允许对以标准方式处理的口腔黏膜存档材料中的基因表达进行定量。
