筛选LPS诱导下THP-1细胞稳定内参基因的研究.docVIP

  筛选 LPS 诱导下 THP-1 细胞稳定内参基因 的研究# 5 10  摘要： 目的 筛选 LPS 诱导 THP-1 细胞前后稳定的内参基因。 方法 运用 RT-qPCR 方法， 分析 10 个候选内参基因（ACTB、GAPDH、RPL37A、PPIB、PGK1、PPIA、SDHA、TBP、 HPRT1、 RPL13A ）在 LPS 刺激 THP-1 细胞前后不同时间的表达稳定性。 结果 经 NormFinder 和 GeNorm 软件分析可知，GAPDH、PGK1 表达稳定，可用来准确校正定量结 果；后期实验中同时选用 GAPDH、PGK1 有助于得到更可靠的结果。结论 RT-qPCR 实验 应该根据细胞和组织的类型及不同实验条件选择最合适的内参基因；选择 2 个或 2 个以上的 内参基因将有助于得到更可靠的结果。 关键词：RT-PCR；内参基因；LPS；THP-1 细胞；NormFinder 软件；GeNorm 软件 15 Gene expression results in lipopolysaccharide-stimulated THP-1 cells depend significantly on the choice of reference  20 25 30 35 40 genes LIANG Junhong1, CAO Ximei2, WAN Dongfang1, ZHANG Chao1, MENG Xiaoping1, GUO Dawei1 (1. Department of Forensic Science, Shanxi Medical University, TaiYuan 030001; 2. Department of Histology and Embryology, Shanxi Medical University, TaiYuan 030001) Abstract: The choice of internal control genes is important since it may affect the study outcome in RT-qPCR. Indeed, it is well-known that expression levels of traditional reference genes can vary across tissue types and across experimental settings within one specific tissue type. The aim of this study is an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in vitro different cultured cells, THP-1. The transcriptional stability of ten potential reference genes (ACTB,GAPDH,RPL37A,PPIB,PGK1,PPIA,SDHATBP, HPRT1 and RPL13A) were evaluated using RT-qPCR and were compared in different treatment, that was un-stimulated or LPS-stimulated cells. The raw Ct values were determined for each candidate gene at different time points following LPS-stimulated or unstimulated cells. Furthermore, all data were analyzed by the geNorm and NormFinder validation programs. Results indicated that DAPDH and PGK1 were the most stable internal control genes in this study. This study provides the comprehensive reported assessment of internal control genes for use in expression studies in vitro cultured cells. These findings further emphasize