人前列腺特异性抗原mRNA实时荧光PCR定量标准品的构建

Construction of the standards for detecting human prostate specific antigen mRNA with real-time fluorescence quantitative polymerase chain reaction

目的构建实时荧光定量PCR(FQPCR)的标准品以定量检测人前列腺特异性抗原(PSA)mRNA的表达。方法以Trizol裂解抽提法提取LNCaP细胞的总RNA。以RTPCR法制备PSAcDNA目的片段并进行扩增;以AT载体克隆法制备重组质粒,并转化E.coliDH5α菌提取重组质粒,联合用氨苄青霉素筛选法和α筛选法、EcoRⅠ限制性酶切和序列分析法鉴定特异性重组质粒。用聚乙二醇沉淀法提纯质粒并检测λ260nm吸光度,确定原液的重组质粒的拷贝浓度并以此制备FQPCR梯度标准品。结果PSAcDNA目的片段成功制备并获得稳定的重组质粒,保持了目的片段的特异性和序列的完整性。结论成功构建了实时荧光定量PCR标准品。

Objective To construct a series of standards for detecting human prostate specific antigen mRNA absolutely with real-time fluorescence quantitative polymerase chai＇a reaction（FQ-PCR）. Methods A pair of primers were designed to amplify a fragment of 145bp between exon2 and exon3, A TaqMan probe modified with 6-Fam at 5-end and Tamra at its 3-end was designed, then, the detection was developed, the PCR product was cloned and sequenced with Beckman CEQ8000. Results The quantitative assay was developed using an external standard reference curve and the PCR product was cloned successfully. Conclusion A series of standards for real-time FQ-PCR analysis have been constructed successfully.