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  • 倪晨明,倪灿荣,金钢,焦莉娟,李连峰,郑建明.胰腺癌石蜡包埋组织中微RNA原位杂交检测技术的优化[J].第二军医大学学报,2018,39(12):1375-1380    [点击复制]
  • NI Chen-ming,NI Can-rong,JIN Gang,JIAO Li-juan,LI Lian-feng,ZHENG Jian-ming.Optimization of in situ hybridization for detecting microRNA in paraffin-embedded pancreatic cancer tissue sections[J].Acad J Sec Mil Med Univ,2018,39(12):1375-1380   [点击复制]
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胰腺癌石蜡包埋组织中微RNA原位杂交检测技术的优化
倪晨明1,倪灿荣2,金钢1*,焦莉娟2,李连峰2,郑建明2*
0
(1. 海军军医大学(第二军医大学)长海医院胰腺外科, 上海 200433;
2. 海军军医大学(第二军医大学)长海医院病理科, 上海 200433
*通信作者)
摘要:
目的 对采用原位杂交检测10%中性甲醛溶液固定的石蜡包埋(FFPE)胰腺癌组织切片中微RNA(miR)的方法进行技术优化,以提高miR的阳性检出率。方法 将20例胰腺癌组织标本构建成组织芯片,使用锁核酸(LNA)标记的探针和显色原位杂交技术检测miR-21和miR-34a的表达。杂交温度分别设为48℃、53℃、56℃,杂交后采用3种不同的洗涤条件(温度、时间和3种不同洗涤缓冲液)进行杂交条件优化。将126例胰腺癌组织标本制成组织芯片,采用优化的显色原位杂交技术检测miR-21和miR-34a的表达。结果 miR-21探针最佳杂交条件为杂交温度53℃下杂交6 h,使用洗涤缓冲液Ⅲ在65℃下洗涤6 min,再用PBS洗涤1 min;miR-34a探针最佳杂交条件为杂交温度53℃下杂交6 h,使用洗涤缓冲液Ⅲ分别在4℃和65℃下洗涤6 min,再用PBS洗涤1 min。在126例胰腺癌组织中,84例(66.7%)miR-21表达阳性,77例(61.1%)miR-34a表达阳性。结论 在FFPE胰腺癌组织切片中,miR-21和miR-34a的最佳杂交温度均为53℃,选择合适的杂交后洗涤温度和洗涤缓冲液有利于提高miR的阳性检出率。
关键词:  原位杂交  微RNA  组织微阵列  胰腺肿瘤  杂交温度
DOI:10.16781/j.0258-879x.2018.12.1375
投稿时间:2018-09-16修订日期:2018-12-07
基金项目:国家自然科学基金(81172077),上海市卫生计生系统重要薄弱学科建设项目(2015ZB0202).
Optimization of in situ hybridization for detecting microRNA in paraffin-embedded pancreatic cancer tissue sections
NI Chen-ming1,NI Can-rong2,JIN Gang1*,JIAO Li-juan2,LI Lian-feng2,ZHENG Jian-ming2*
(1. Department of Pancreatic Surgery, Changhai Hospital, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Pathology, Changhai Hospital, Navy Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding authors)
Abstract:
Objective To optimize in situ hybridization (ISH) method for microRNA (miR) detection in formalin-fixed and paraffin-embedded (FFPE) pancreatic cancer tissue sections, so as to improve the sensitivity of miR detection. Methods Tissue microarray (TMA) was constructed from 20 pancreatic cancer tissue specimens. Using locked nucleic acid (LNA) labeled probe, we examined the expression levels of miR-21 and miR-34a by chromogenic in situ hybridization (CISH). The hybridization temperatures were set at 48, 53 and 56℃, respectively. Three different washing approaches (temperature, time and 3 different washing buffers) were adopted to optimize the hybridization conditions. TMA was prepared using 126 pancreatic cancer tissue specimens, and optimal CISH was used to detect the expression levels of miR-21 and miR-34a. Results The optimal hybridization conditions of miR-21 probe were as follows:hybridization at 53℃ for 6 h, washing with washing buffer Ⅲ at 65℃ for 6 min, and then washing with PBS for 1 min. The optimal hybridization conditions of miR-34a probe were as follows:hybridization at 53℃ for 6 h, washing with washing buffer Ⅲ at 4℃ and 65℃ for 6 min, respectively, and then washing with PBS for 1 min. Of 126 pancreatic cancer tissue specimens, 84 (66.7%) had positive expression of miR-21, and 77 (61.1%) had positive expression of miR-34a. Conclusion The optimal hybridization temperatures of miR-21 and miR-34a are both 53℃ in the FFPE pancreatic cancer tissue sections, and appropriate washing temperature and washing buffer after hybridization are conducive to improve the positive detection rate of miR.
Key words:  in situ hybridization  microRNAs  tissue microarray  pancreatic neoplasms  hybridization temperature