定量PCR分析人肺癌细胞NCI-H1299基因表达的内参基因选择

Comparison of the Reference Genes for Gene Expression Studies in Human Lung Cancer Cell Line NCI-H1299 by Real-time Quantitative PCR

目的评价实时荧光定量PCR分析人肺癌细胞NCI-H1299中Merm1/Wbscr22基因表达时6个候选内参基因表达的稳定性,筛选最适的内参基因。方法选择ACTB、B2M、GAPD、HPRT1、RPL32及TBP作为候选内参基因,利用geNorm,NormFinder及RefFinder程序分析实时荧光定量PCR数据,评价6个候选内参基因在NCI-H1299细胞株中的表达稳定性,筛选最适内参基因,同时观察选用不同内参基因对目的基因相对表达量分析的影响。结果 6个候选内参基因在NCI-H1299细胞株中的表达稳定性由强至弱排序为:RPL32>HPRT1>GAPD>ACTB>TBP>B2M,选择不同内参基因影响目的基因Merm1/Wb-scr22的相对表达量。结论 RPL32+HPRT1是实时荧光定量PCR分析人肺癌细胞NCI-H1299中Merm1/Wbscr22基因表达的最适内参基因组合。

Objective To compare and select the most consistent reference gene（s） used in real - time quantitative PCR analysis of Merml/Wbscr22 mRNA expression level in human lung cancer cell line NCI - H1299. Methods A total of six genes, namely ACTB（beta actin） , B2M （ beta - 2 - microglobulin） , GAPD （ glyceraldehyde - 3 - phosphate dehydrogenase） , HPRT1 （ Hypoxanthinc phosphori-bosyl transferase 1 ）, RPL32 （ ribosomal protein L32） and TBP（TATA - binding protein）, were chosen as the candidate reference genes. Theirs expression stability were estimated by three programs geNorm, NormFinder and RefFinder. The effects of different reference genes on the relative expression level of the target gene Merml/Wbscr22 were analyzed. Results The expression consistency of the 6 genes was （from high to low） ： RPL32 〉 HPRT1 〉 GAPD 〉 ACTB 〉 TBP 〉 B2M. The relative expression level of Merml/Wbser22 mRNA was altered using different reference gene（s）. Conclusion RPL32 and HPRTlwere the most preferable combination of the reference genes for real-time quantitative PCR analysis of Merml/Wbscr22 mRNA level in NCI- H1299 cells.