石蜡包埋乳腺癌组织的RNA提取及基因表达研究  

RNA extraction and gene expression of paraffin-embedded breast cancer samples

作　　者：孙冰[1] 郭晓红[2] 张利利[3] 张峰[3] 吴世凯[1] 宋三泰[1] 江泽飞[1] 王敦梅[3] 苏敏[2] 

机构地区：[1]军事医学科学院附属医院,北京100071 [2]汕头大学医学院 [3]中国科学院,北京基因组研究所

出　　处：《山东医药》2009年第12期15-17,共3页Shandong Medical Journal

摘　　要：目的探索从石蜡包埋乳腺癌样本中提取总RNA的质量和产量，并检测其基因的表达情况。方法收集保存时间1—17a不等的人乳腺癌石蜡组织样本153例，取5um厚切片1—15张，用Roche和Ambion两种试剂盒提取总RNA。采用随机引物将mRNA反转录为eDNA，SYBRGreen Ⅰ染料法实时荧光定量PCR分析ACTB、GAPDH、RPLPO和GUSB管家基因mRNA的表达情况。结果153例标本总RNA浓度在3—375ng／ul范围，总产量0．18—18．5ug，A260／280值在1．32—2．07范围，RNA琼脂糖凝胶电泳片段弥散在100—1000bp之间。81例样本的4个管家基因的平均Ct值在24—30范围，随着样本保存时间的延长，Ct值也逐渐增加。结论从石蜡包埋样品标本中提取的RNA产量及质量可满足实验的要求，总RNA有所降解，但可以进行目的基因mRNA的检测，保存10a以上的样本也能够进行基因表达研究，在基础研究和临床实践方面具有广阔的应用价值。Objective To study the quality and yield of total RNA extracted from paraffin-embedded breast cancer samples and their gene expression. Methods 153 cases of paraffin-embedded human breast cancer samples were collected with storage time from 1 to 17 years. Total RNA was extracted by using two commercial extraction kit from 1 - 15 slices of 5um in thickness, mRNAs were reversely transcripted into cDNA with random primers, and four different housekeeping genes in mRNA level were detected using SYBR Green Ⅰ Quantitative Real-time PCR. Results Of all the 153 samples, total RNA ranged from 3 to 375 ng/ul in concentrations, 0.18 - 18.5 ug in total yield, 1.32 - 2.07 in A260/280. Fragments dispersed between 100 - 1 000 bp in RNA agarose gel electrophoresis. The expression of four housekeeping genes in 81 cases ranged from 24 to 30 in Ct value on average, and Ct value gradually increased with the extension of storage time of samples. Conclusion The RNA extracted from FFPE specimens can meet the requirements of the experiment. The detection of target gene in mRNA level can be carried out with the degraded total RNA, even in samples stored more than 10 years. It will make a great significance in basic research and clinical practice.

关 键 词：石蜡包埋组织 RNA提取 实时逆转录聚合酶链反应 基因表达 

分 类 号：R737.9[医药卫生—肿瘤]