Identification of novel reference genes using multiplatform expression data and their validation for quantitative gene expression analysis.
Normalization of mRNA levels using endogenous reference genes (ERGs) is critical for an accurate comparison of gene expression between different samples. Despite the popularity of traditional ERGs (tERGs) such as GAPDH and ACTB, their expression variability in different tissues or disease status has been reported. Here, we first selected candidate housekeeping genes (HKGs) using human gene expression data from different platforms including EST, SAGE, and microarray, and 13 novel ERGs (nERGs) (ARL8B, CTBP1, CUL1, DIMT1L, FBXW2, GPBP1, LUC7L2, OAZ1, PAPOLA, SPG21, TRIM27, UBQLN1, ZNF207) were further identified from these HKGs. The mean coefficient variation (CV) values of nERGs were significantly lower than those of tERGs and the expression level of most nERGs was relatively lower than high expressing tERGs in all dataset. The higher expression stability and lower expression levels of most nERGs were validated in 108 human samples including formalin-fixed paraffin-embedded (FFPE) tissues, frozen tissues and cell lines, through quantitative real-time RT-PCR (qRT-PCR). Furthermore, the optimal number of nERGs required for accurate normalization was as few as two, while four genes were required when using tERGs in FFPE tissues. Most nERGs identified in this study should be better reference genes than tERGs, based on their higher expression stability and fewer numbers needed for normalization when multiple ERGs are required.
Kwon
Mi Jeong
MJ
Laboratory of Molecular Pathology, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea.
Oh
Ensel
E
Lee
Seungmook
S
Roh
Mi Ra
MR
Kim
Si Eun
SE
Lee
Yangsoon
Y
Choi
Yoon-La
YL
In
Yong-Ho
YH
Park
Taesung
T
Koh
Sang Seok
SS
Shin
Young Kee
YK
D001483:Base Sequence; D017931:DNA Primers; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers