UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines
UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines
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DOI：
10.1007/s10616-011-9383-4
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发表时间：
2011-12-01
期刊：
影响因子：
2.2
通讯作者：
Few, Ling Ling
中科院分区：
文献类型：
--
作者：
Chua, Siang Ling;Too, Wei Cun See;Few, Ling Ling
使用实时PCR的体外基因表达的相对定量需要稳定表达的参考基因用于标准化。本研究从MCF 7、HCT 116和HepG 2细胞中提取总RNA，用试剂盒转化为cDNA，通过实时荧光定量PCR（real-time PCR）分析12个参考基因的表达水平，筛选出最理想的参考基因，用于基因表达研究中的精确标准化。用geNorm和Normalt软件对参比基因的稳定性进行分析，结果显示C（t）值变化范围较大。geNorm分析显示以下基因的稳定性排序：UBC、YWHAZ > RPLP > TBP > ACTB > HPRT 1> PPIA > GAPDH > GUSB > B2 M> TUBB > RRN 18 S。Normandy获得了相似的参考基因排名，并且使用两种方法获得的四个最稳定的参考基因是相同的。基于以上分析，UBC和YWHAZ被认为是最合适的两个参考基因。为了在正式实验中进一步评估UBC和YWHAZ的稳定性，用5-aza-dC和TSA处理MCF 7、HCT 116和HepG 2细胞系。UBC和YWHAZ在对照组和处理组中均表现出稳定的表达水平。因此，我们建议UBC和YWHAZ是我们使用MCF 7，HCT 116和HepG 2细胞系进行基因表达研究的两个最合适的参考基因。
Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C(t) values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.