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.

Jing Cai,Hui Ding,Man Xiao,Bangxing Huang,Ling Liu,Weihong Dong,Tao Li,Henghui Cheng,Zehua Wang

doi:10.1371/journal.pone.0095974

Jing Cai, Hui Ding + Show 7 more

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https://doi.org/10.1371/journal.pone.0095974

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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.

Highlights

Despite advances in the understanding of potential biomarkers and therapeutic targets, effective screening techniques and therapies of ovarian cancer remain a challenging task, partly due to tissue heterogeneity
Formalin-fixed, paraffin-embedded tissues (FFPE), which represent an alternative for nucleic acids extraction and downstream experiments, such as quantitative real-time PCR, are more widely available in clinical laboratories
Results showed that epithelial cells only occupied a small percentage of the whole ovarian tissues, especially the normal ovary

Summary

Introduction

Despite advances in the understanding of potential biomarkers and therapeutic targets, effective screening techniques and therapies of ovarian cancer remain a challenging task, partly due to tissue heterogeneity. Numerous studies have analyzed genetic alterations or gene expression profiles in ovarian cancer by making the comparison between cancerous tissue and normal ovarian tissue. The dissected tissues, as homogeneous starting materials, can be used for a variety of analyses, such as transcriptomic and proteomic studies [5]. During this process, the use of Rnase-free reagents and cresyl violet staining are recommended to minimize RNA degradation and maintain tissue morphology, respectively [6,7]. Fresh cryosections are preferred for nucleic acids extraction using LMD, fresh sections are usually scarce in basic and clinical research. Formalin-fixed, paraffin-embedded tissues (FFPE), which represent an alternative for nucleic acids extraction and downstream experiments, such as quantitative real-time PCR (qPCR), are more widely available in clinical laboratories

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