碱基序列标记法结合焦磷酸测序测定不同来源基因表达量

Differential Gene Expression Analysis by Combining Sequence-Tagged Reverse-Transcription Polymerase Chain Reaction with Pyrosequencing

建立了一种将序列标记反转录聚合酶链反应(PCR)与焦磷酸测序技术结合的相对基因表达量测定法(简称"SRPP")。先用来源特异性引物对不同来源的同一基因通过反转录标记上特异性标签,PCR后用焦磷酸测序法对扩增产物进行序列解码,使得测序结果中的序列代表基因的来源,峰高代表基因在不同来源中的相对表达量。用实时荧光定量PCR法对本方法的准确性进行了验证,结果表明,SRPP可以同时准确测定同一基因在3个不同来源中的表达量,并实际测定了Egr1基因在糖尿病、肥胖和正常小鼠肝中的表达量差异。

It is an important way to understand gene function by relatively comparing gene expression levels among different tissues or cells. For the moment, most of the methods for gene expression detection are based on dye labels. To establish a novel approach without the use of a dye label, a sequence-tagged reverse transcription polymerase chain reaction（PCR） coupled with pyrosequencing （SRPP for short） was proposed. In this technique, the gene at a source was labeled with a source-specific sequence by sequence-tagged reverse transcription. Then PCR on the pools of each source-specific RT product was performed, and the sourcespecific amplicons were decoded by pyrosequencing. In the pyrogram, the sequence represents the gene source, and the peak intensity represents the relative expression level of the gene in the corresponding source. The accuracy of SRPP was confirmed by real-time quantitative PCR. Finally the relative expression levels of the Egrl gene among the diabetes model mice, obesity model mice and normal mice were successfully detected. In comparison with real-time PCR, the advantages of SRPP include without dye, inexpensive instruments, and simultaneous comparison of a given gene expressed in multiple sources.