Using endogenous control genes in real-time RT-PCR

Gene Expression Web
QuantiTect Primer Assays as endogenous controls
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When performing relative quantification of the expression of a target gene, it is important to choose a suitable gene for use as a reference or endogenous control. An endogenous control gene is a gene whose expression level should not differ between samples, such as a housekeeping or maintenance gene. Comparison of the CT value of a target gene with that of the endogenous control gene allows the gene expression level of the target gene to be normalized to the amount of input RNA or cDNA. This is done without determining the exact amount of template used in the reaction. The use of an endogenous control gene corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recovery, and differences in sample handling. Genes commonly used as references are shown in the tables below.
Relative Expression Levels of Commonly Used Human Housekeeping Genes 
Relative Expression Levels of Commonly Used Mouse Housekeeping Genes
Relative expression levels of commonly used human housekeeping genes
Gene symbol Gene name  Relative expression level*
 RRN18S 18S ribosomal RNA  ++++
 ACTB  Actin, beta  +++
 GAPD  Glyceraldehyde-3-phosphate dehydrogenase  +++
 PGK1  Phosphoglycerate kinase 1  +++
 PPIA  Peptidylprolyl isomerase A (cyclophilin A)  +++
 RPL13A  Ribosomal protein L13a  +++
 RPLP0  Ribosomal protein, large, P0  +++
 B2M  Beta-2-microglobulin  ++ – +++
 YWHAZ  Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,  zeta polypeptide  ++ – +++
 SDHA  Succinate dehydrogenase  ++
 TFRC  Transferrin receptor (p90, CD71)  ++
 ALAS1  Aminolevulinate, delta-, synthase 1  +
 GUSB  Glucuronidase, beta  +
 HMBS  Hydroxymethyl-bilane synthase  +
 HPRT1  Hypoxanthine phosphoribosyltransferase 1  +
 TBP  TATA box binding protein  +
 TUBB  Tubulin, beta polypeptide  +
* "+" indicates relative abundance of the transcripts.

Relative expression levels of commonly used mouse housekeeping genes
Gene symbol Gene name  Relative expression level*
 Rn18s  18S ribosomal RNA  ++++
 Actb  Actin, beta, cytoplasmic  +++
 Gapd  Glyceraldehyde-3-phosphate dehydrogenase  +++
 Ppia  Peptidylprolyl isomerase A (cyclophilin A)  +++
 Rpl13a  Ribosomal protein L13a  +++
 Arbp  Acidic ribosomal phosphoprotein PO  +++
 B2m  Beta-2-microglobulin  ++ –+++
 Hmbs  Hydroxymethylbilane synthase  ++ –+++
 Pgk1  Phosphoglycerate kinase 2  ++
 Alas1  Aminolevulinic acid synthase 1  +
 Gus-b  Glucuronidase, beta  +
 Hprt  Hypoxanthine guanine phosphoribosyl transferase  +
 Sdha  Succinate dehydrogenase  +
 Tbp  TATA box binding protein  +
 Trfr  Transferrin receptor  +
 Tubb4  Tubulin, beta 4  +
 Ywhaz  Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide  +
* "+" indicates relative abundance of the transcripts.


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