关键词: 反转录巢式多重聚合酶链式反应, 时钟基因, 单细胞, NIH/3T3

Abstract:

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.

Key words: multiplex nested RT-PCR, clock gene, single cell, NIH/3T3