A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas.
A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas.
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发表时间：
1993-03
期刊：
影响因子：
11.2
通讯作者：
R. Radinsky;C. Bucana;L. Ellis;R. Sanchez;K. Cleary;D. Brigati;I. Fidler
中科院分区：
文献类型：
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作者：
R. Radinsky;C. Bucana;L. Ellis;R. Sanchez;K. Cleary;D. Brigati;I. Fidler
我们开发了一种快速比色原位mRNA杂交方法来分析石蜡包埋的人结肠癌手术标本中的表皮生长因子受体（EGF-R）转录物。该技术基于使用24碱基寡核苷酸探针，在3'端标记6个生物素分子。使用高生物素化的30个残基长的脱氧胸腺苷寡核苷酸探针验证mRNA的完整性，并使用标记的egf -r特异性正义和反义探针证实该反应的特异性。在Microprobe系统中使用的亲和素碱性磷酸酶检测和毛细管技术允许在5小时内完成该过程。在培养中生长并用福尔马林固定的人A431表皮样癌细胞以及裸鼠肿瘤生长s.c.的石蜡包埋切片作为阳性对照。用反义EGF-R寡核苷酸探针原位杂交，分别通过Northern blot和免疫组织化学观察到与EGF-R mRNA和蛋白水平直接相关的水平。石蜡包埋的人原发性结肠癌和肝脏及淋巴结转移灶切片的原位杂交显示，EGF-R反义寡核苷酸探针对细胞进行特异性染色，与Northern blot和免疫组织化学分析直接相关。由于这种快速和敏感的原位mRNA杂交技术可以用于适当保存的石蜡包埋组织，因此它允许使用档案材料对人类肿瘤标本进行回顾性分析。
We have developed a rapid colorimetric in situ mRNA hybridization procedure to analyze epidermal growth factor receptor (EGF-R) transcripts in paraffin-embedded surgical specimens of human colon carcinomas. This technique is based on the use of 24-base oligonucleotide probes labeled with 6 biotin molecules at the 3' end. mRNA integrity was verified using a hyperbiotinylated 30-residue-long deoxythymidylate oligonucleotide probe, and the specificity of the reaction was confirmed by using labeled EGF-R-specific sense and antisense probes. Avidin alkaline phosphatase detection and the capillary technology used in the Microprobe System allowed for completion of the procedure in under 5 h. The human A431 epidermoid carcinoma cells growing in culture and fixed with formalin as well as paraffin-embedded sections of this tumor growing s.c. in nude mice served as positive controls. In situ hybridization with antisense EGF-R oligonucleotide probes directly correlated with EGF-R mRNA and protein levels observed by Northern blot and immunohistochemistry, respectively. In situ hybridization of paraffin-embedded sections of primary human colon carcinoma and metastases from liver and lymph node revealed cell-specific staining with EGF-R antisense oligonucleotide probes that correlated directly with Northern blot and immunohistochemistry analyses. Since this rapid and sensitive in situ mRNA hybridization technique can be used in properly preserved paraffin-embedded tissue, it allows for retrospective analyses of human tumor specimens using archival material.