Selection of Housekeeping Genes for Real-time Fluorescence Quantitative RT-PCR in Skin of Fine-wool Sheep

摘要

At present, transcription analysis of gene expression commonly uses a single housekeeping gene as control for normalization. The levels of six housekeeping genes (including 18 SrRNA, GAPDH, ACTB, RPL13A, B2M and TBP) in the skin tissue of fine-wool sheep were estimated by SYBR Green I which belongs to a method of real-time fluorescence quantitative RT-PCR expression. The results showed that differences in expression levels were observed by analysis of geNorm program, an optimal number of control genes for normalization in skin tissue of fine-wool sheep would be 3 and 18 SrRNA, GAPDH, ACTB were finally determined as suitable internal control genes with the results of V2/3(0.163)>0.15, while V3/4(0.100)<0.15 and the M value B2M>RPL13A>TBP> 18 SrRNA>ACTB(GAPDH). The results of this study revealed using three genes (ACTB, GAPDH and 18 SrRNA) in experimental system about sheep, their mRNA expression levels would not change apparently were found. The significance of this study provided convincing references and methodology for selection of housekeeping genes and normalization in gene expression analysis with RT-PCR. In conclusion, the necessity of choosing reference genes was proved and a good way was introduced to select reference genes when experiments were handled by different empirical factors (especially under the effect of new materials). This approach might be useful for studying on the candidate genes which associated with the wool fineness.