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Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.

Romani C, Calza S, Todeschini P, Tassi RA, Zanotti L, Bandiera E, Sartori E, Pecorelli S, Ravaggi A, Santin AD, Bignotti E - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.

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.

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Related in: MedlinePlus

Expression levels of candidate reference genes in malignant (hatched boxes) and non-malignat (open boxes) endometrial samples.Values are given as calibrated normalized relative quantities (CNRQ). Boxes indicate IQR for the data of unmatched samples in each group.
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pone-0113781-g001: Expression levels of candidate reference genes in malignant (hatched boxes) and non-malignat (open boxes) endometrial samples.Values are given as calibrated normalized relative quantities (CNRQ). Boxes indicate IQR for the data of unmatched samples in each group.

Mentions: Data on non-malignant and malignant samples are separately shown as box plot in Fig. 1. All reference genes showed a substantial higher spread in malignant samples compared to non-malignant ones. The genes showing the higher Interquatile-range (IQR) were B2M (IQR = 0.402) and ACTB (IQR = 0.295) while PPIA, POLR2A and HPRT1 show the smallest IQR (0.153, 0.187 and 0.197 respectively).


Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.

Romani C, Calza S, Todeschini P, Tassi RA, Zanotti L, Bandiera E, Sartori E, Pecorelli S, Ravaggi A, Santin AD, Bignotti E - PLoS ONE (2014)

Expression levels of candidate reference genes in malignant (hatched boxes) and non-malignat (open boxes) endometrial samples.Values are given as calibrated normalized relative quantities (CNRQ). Boxes indicate IQR for the data of unmatched samples in each group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256201&req=5

pone-0113781-g001: Expression levels of candidate reference genes in malignant (hatched boxes) and non-malignat (open boxes) endometrial samples.Values are given as calibrated normalized relative quantities (CNRQ). Boxes indicate IQR for the data of unmatched samples in each group.
Mentions: Data on non-malignant and malignant samples are separately shown as box plot in Fig. 1. All reference genes showed a substantial higher spread in malignant samples compared to non-malignant ones. The genes showing the higher Interquatile-range (IQR) were B2M (IQR = 0.402) and ACTB (IQR = 0.295) while PPIA, POLR2A and HPRT1 show the smallest IQR (0.153, 0.187 and 0.197 respectively).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: "Angelo Nocivelli" Institute of Molecular Medicine, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.

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.

Show MeSH
Related in: MedlinePlus