反转录巢式多重聚合酶链式反应体系检测单细胞中时钟基因表达

Analysis of key clock genes expression in individual cells with multiplex nested reverse transcriptase-PCR

目的建立一种高度灵敏的能够在单细胞水平检测时钟基因表达的反转录巢式多重聚合酶链式反应(multiplex nestedreverse transcription-polymerase chain reaction,multiplex nested RT-PCR)体系。方法选取核心时钟基因bmal1,clock,per1,per2,cry1和cry2,以及看家基因β-actin,分别设计和优化巢式引物对。以基因质粒为模板,检测巢式多重PCR体系的灵敏度。体外转录得到各个基因的RNA模板,并检测反转录巢式多重PCR体系的灵敏度。使用手动微操作器,显微镜下负压获取悬浮单个NIH/3T3细胞,使用反转录巢式多重PCR体系检测其中目的时钟基因。应用实时定量PCR检测整体水平时钟基因表达情况,与单细胞水平进行比较。结果巢式多重PCR检测体系可以检测出单拷贝质粒DNA。反转录巢式多重PCR能够检测出4拷贝RNA模板。利用上述体系发现,NIH/3T3单细胞中时钟基因环路表达存在很大差异,同时发现群体水平per1和per2表达的高峰和低谷间差异有统计学意义,而单细胞水平per1表达则差异无统计学意义。结论建立了高度灵敏的用于检测时钟基因表达的反转录巢式多重PCR体系,揭示了单细胞水平时钟基因表达的异质性,也验证了整体水平与单细胞水平时钟基因表达的差异。

Objective To establish a highly sensitive multiplex nested reverse transcriptase polymerase chain reaction（RT-PCR） system for analysis of key clock genes expression in individual cells.Methods Six key clock genes（bmal1,clock,per1,per2,cry1,cry2） and a housekeeping gene（β-actin） were chosen and the nested primer pairs were designed using Oligo 6.0 primer analysis software.Sensitivity for multiplex nested PCR and RT-PCR was examined separately.And then the expression profiles of clock gene were examined in single cells collected with micromanipulator.Real-time PCR was used to test the general level of the clock gene expression.And then the clock gene expression profiles were compared between general level and single cell level.Results One DNA copy of each gene could be detected with our multiplex nested PCR system.Four RNA copies of each gene could be detected with the multiplex nested RT-PCR system.The expression pattern of clock genes was distinct among individual NIH/3T3 cells.The expression pattern of per1 and per2 were distinct in general level.But the expression pattern of per1 was not distinct in single cell.Conclusion A highly sensitive multiplex nested RT-PCR system examining clock genes was established.Molecular clock is poorly synchronized among individual NIH/3T3 cells,it was also proved that the expression pattern of clock gene was distinct between general level and single cell.