Back to Search
Start Over
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
- Source :
- PLoS ONE, PLoS ONE, Vol 9, Iss 4, p e95974 (2014)
- Publication Year :
- 2014
-
Abstract
- 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.
- Subjects :
- Adult
Candidate gene
Tissue Fixation
Tumor Physiology
lcsh:Medicine
Gene Expression
Biology
Carcinoma, Ovarian Epithelial
Real-Time Polymerase Chain Reaction
Reference genes
Formaldehyde
Gene expression
Molecular Cell Biology
Basic Cancer Research
Genetics
Medicine and Health Sciences
Humans
Neoplasms, Glandular and Epithelial
lcsh:Science
Microdissection
Laser capture microdissection
Regulation of gene expression
Ovarian Neoplasms
Multidisciplinary
Genes, Essential
Paraffin Embedding
Lasers
lcsh:R
Ovary
Biology and Life Sciences
Cancers and Neoplasms
Epithelial Cells
Cell Biology
Middle Aged
Reference Standards
Molecular biology
Housekeeping gene
Ovarian Cancer
Gene Expression Regulation, Neoplastic
Real-time polymerase chain reaction
Oncology
lcsh:Q
Female
Oncology Agents
Gynecological Tumors
Research Article
Subjects
Details
- ISSN :
- 19326203
- Volume :
- 9
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- PloS one
- Accession number :
- edsair.doi.dedup.....b13b74a4d4b0720f7b97c2ba3aedc93c