title: Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.
abstract: Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Romani
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Stefano
authors.initials: S
authors.lastname: Calza
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Todeschini
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Renata A
authors.initials: RA
authors.lastname: Tassi
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Zanotti
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Bandiera
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Sartori
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Pecorelli
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Ravaggi
authors.affiliation: Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Alessandro D
authors.initials: AD
authors.lastname: Santin
authors.affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
authors.forename: Eliana
authors.initials: E
authors.lastname: Bignotti
mesh_terms: D000199:Actins / Q000235:genetics / Q000592:standards; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018269:Carcinoma, Endometrioid / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015331:Cohort Studies; D004717:Endometrium / Q000378:metabolism / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics / Q000592:standards; D006801:Humans; D008875:Middle Aged; D009367:Neoplasm Staging; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012016:Reference Values; D012984:Software
chemical_list: D000199:Actins; D012337:RNA, Ribosomal, 18S; D012313:RNA; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)