The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.
Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Jing
J
Cai
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Tao
T
Li
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Bangxing
B
Huang
Department of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Henghui
H
Cheng
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Hui
H
Ding
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Weihong
W
Dong
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Man
M
Xiao
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Ling
L
Liu
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Zehua
Z
Wang
D000328:Adult; D000077216:Carcinoma, Ovarian Epithelial; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005557:Formaldehyde / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D020043:Genes, Essential / Q000235:genetics; D006801:Humans; D007834:Lasers; D042282:Microdissection; D008875:Middle Aged; D009375:Neoplasms, Glandular and Epithelial / Q000235:genetics / Q000473:pathology; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D010053:Ovary / Q000166:cytology / Q000473:pathology; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D016707:Tissue Fixation
D005557:Formaldehyde