This work was supported by a grant from The National Natural Sciences Foundation of China (39970270).
为了建立一种适于实验室乃至常规定量检测mRNA表达的、可快速构建cRNA标准曲线的方法,设计带有T7启动子序列和PolyT序列的引物对目的基因和内参照进行PCR,克隆入载体作为体外合成cRNA的模板,快速构建cRNA标准.结果表明:该曲线的线性范围至少达6个数量级,相关系数为0.99.该法快速、简便,适用于所有靶基因.
To establish a rapid construction cRNA standard curves method applicable to common laboratory, PCR was carried to amplify the target gene and internal reference gene using primers containing T7 promotor and polyT sequence, PCR product was cloned as the template of T7 RNase polymerase to synthesize cRNA in vitro for establishing standard curves. The result shows that constructed cRNA curve has a wide linear range of at least six orders and correlation coefficient is 0.99.The rapid and simple method can be applicable to measurement of transcripts for any gene.
张燕,沈茜.一种快速构建cRNA标准曲线检测基因表达方法的建立[J].生物化学与生物物理进展,2003,30(3):474-477 Zhang Yan, Shen Qian. Rapid Construction of cRNA Standard Curves in Real-time Reverse Transcription Polymerase Chain Reaction to Measure mRNA Expression[J]. Progress in Biochemistry and Biophysics,2003,30(3):474-477
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