RT-PCR中加工过的假基因的干扰及解决办法

INTERFERENCE OF HUMAN PROCESSED PSEUDOGENE AND CORRESPONDING COUNTER MEASURES IN REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

目的发现和克服ＲＴ－ＰＣＲ中可能存在的假基因的干扰。方法采用ＲＴ－ＰＣＲ法扩增人肺鳞癌标本中角蛋白１９（ＣＫ１９ｃＤＮＡ或ＤＮＡ序列。结果通过设立分别以基因组ＤＮＡ和未经逆转录的ＲＮＡ为模板的对照可观察到ＣＫ１９假基因的干扰现象。用脱氧核糖核酸酶预处理ＲＮＡ标本或在设计错配引物的基础上选择合适的退火温度可消除干扰。结论在运用ＲＴ－ＰＣＲ法分析哺乳动物基因表达时常存在假基因的干扰。

Objective This study aimed to set up an effective method to find the interference of processed pseudogene and overcome it in RT PCR. Methods Using RT PCR methods, the authors amplified the cytokeratin 19 cDNA or DNA sequence in human lung squamous cell carcinoma samples.Results They could find the influence of pseudogene through performing genomic DNA and nonreversed transcribed RNA controls.By digesting the extracted RNA sample with RNase free DNase or regulating the annealing temperature based on mismatched primers,they could effectively eliminate the interference. Conclusion The presence of processed pseudogene usually interferes in the experimental protocol in RT PCR.This problem can be resolved with the method aforementioned.