Both beta-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer.
BACKGROUND
Beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been frequently considered as constitutive house keeping genes for RT-PCR and used to normalize changes in specific gene expressions. However, these expressions have been shown to be affected by the sample type and experimental conditions. We investigated which housekeeping gene is useful to study gene expression of paraffin embedded human tissue samples of prostate cancer.


METHODS
Fifteen pairs of cancer and corresponding normal tissue were obtained from patients with prostate cancer. We evaluated gene expression of beta-actin, GAPDH, androgen receptor (AR), and heat-shock 70-kd protein 5 (HSPA5) using laser captured microdissection and quantitative RT-PCR. AR and HSPA5 gene expression were normalized to each of these reference genes using the 2(-DeltaDeltaCt) method of relative quantification. The quantity 2(Ct(normal)-Ct(cancer)) divided by ratio of cDNA(cancer)/cDNA (normal) was used for comparing differences between cancer and normal tissue in GAPDH and beta-actin expression.


RESULTS
Ct value of beta-actin was significantly correlated with that of GAPDH (r = 0.443, P = 0.014). AR and HSPA5 gene expression levels using beta-actin for normalization were significantly correlated with these gene expression levels using GAPDH (AR; r = 0.689, P = 0.004, HSPA5; r = 0.879, P < 0.001). Both reference genes were expressed more highly in cancer tissue than in normal tissue, with that of GAPDH being significantly different between cancer tissue and normal tissue (P = 0.029).


CONCLUSIONS
The good correlation between gene expression values obtained when using beta-actin and GAPDH as reference genes suggests that either gene is a valid denominator for gene expression studies in prostate cancer.
Mori
Ryutaro
R
Department of Biochemistry & Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA.
Wang
Qingcai
Q
Danenberg
Kathleen D
KD
Pinski
Jacek K
JK
Danenberg
Peter V
PV
D000199:Actins / Q000096:biosynthesis / Q000235:genetics*; D015331:Cohort Studies; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D020869:Gene Expression Profiling; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D011944:Receptors, Androgen / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D018709:Statistics, Nonparametric
D000199:Actins; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D012334:RNA, Neoplasm; D011944:Receptors, Androgen; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases