Reference Genes and Controls

Reference genes and controls for gene expression analysis

For relative quantification of gene expression, it is important to choose a suitable gene to use as a reference (see table Housekeeping genes commonly used as endogenous references). The expression level of the reference gene must not vary under experimental conditions, or in different states of the same tissue or cell line (e.g., “disease” versus “normal” samples). The expression level of the reference RNA should also be approximately the same as the RNA under study. Reference RNA commonly used for relative quantification includes b-actin, b-2-microglobulin, peptidylprolyl isomerase A, and GAPDH mRNAs, and also 18S rRNA. b-actin mRNA is ubiquitously expressed and was one of the first RNAs to be used as a reference sequence. However, its transcription levels may vary and the presence of pseudogenes may mean that genomic DNA is detected during real-time PCR, leading to inaccuracies in quantification. GAPDH is a housekeeping gene commonly used as a reference for quantification of gene expression. GAPDH mRNA levels may vary between individuals, at different stages of the cell cycle, and following treatment with different drugs, making GAPDH unsuitable as a reference in some systems. As 18S rRNA is not an mRNA, its expression levels in the cell may not accurately reflect the cellular mRNA population. Therefore, a combination of genes may provide the most reliable reference for quantification studies.
Housekeeping genes commonly used as endogenous references
Gene Human gene symbol Mouse gene symbol Relative expression
level in humans*  
Relative expression
level in mice*
18S ribosomal RNA RRN18S Rn18s ++++ ++++
Actin, beta ACTB  Actb +++ +++
GAPDH Gapdh +++ +++
Phosphoglycerate kinase 1 PGK1 Pgk1 +++ +++
Peptidylprolyl isomerase A PPIA Ppia +++ +++
Ribosomal protein L13a RPL13A Rpl13a +++ +++
Ribosomal protein, large, P0 RPLP0 +++
Acidic ribosomal
phosphoprotein PO
Arbp +++
Beta-2-microglobulin B2M B2m ++ to +++ ++ to +++
Tyrosine 3-monooxygenase/
activation protein,
zeta polypeptide
YWHAZ Ywhaz ++ to +++ +
Succinate dehydrogenase
complex, subunit A,
flavoprotein (Fp)
SDHA Sdha ++ +
Transferrin receptor TFRC Tfrc ++ +
Aminolevulinate, delta-,
synthase 1
ALAS1 Alas1 + +
Glucuronidase, beta GUSB Gusb + +
HMBS Hmbs + ++ to +++
phosphoribosyltransferase 1
HPRT1 Hrpt1 + +
TATA box binding protein TBP Tbp + +
Tubulin, beta TUBB +
Tubulin, beta 4 Tubb4 +