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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

Selection of the most suitable reference genes for normalization in endometrial cancer samples using geNorm analysis.Results are presented according to the output file of the geNorm program [11]. (A) Average expression stability value (M) of control genes calculated by a stepwise exclusion of the least stable genes: the program enables elimination of the worst-scoring gene with the highest M value and recalculation of new M value for the remaining genes. X axis from the left to the right indicates gene rank according to expression stability, while Y axis indicate the stability measure M. (B) Determination of the optimal number of control genes for normalization.
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pone-0113781-g002: Selection of the most suitable reference genes for normalization in endometrial cancer samples using geNorm analysis.Results are presented according to the output file of the geNorm program [11]. (A) Average expression stability value (M) of control genes calculated by a stepwise exclusion of the least stable genes: the program enables elimination of the worst-scoring gene with the highest M value and recalculation of new M value for the remaining genes. X axis from the left to the right indicates gene rank according to expression stability, while Y axis indicate the stability measure M. (B) Determination of the optimal number of control genes for normalization.

Mentions: From the theoretical point of view, genes whose expression are equivalent between malignant and non malignant tissue samples are all suitable reference for relative quantification of target genes in gene profiling studies. Thus, according to the statistical analysis performed on the expression level of the 10 selected reference genes, GUSB, HPRT1 and PPIA were the best candidate for normalization purposes in gene expression studies in endometrial cancer versus normal tissue. To validate and confirm this result, all 10 reference genes including the best performing GUSB, HPRT1 and PPIA were included in the program geNorm which calculated the gene expression stability measure M of one gene based on the average pairwise variation between all studied genes. The lowest M value characterized the gene with the most stable expression. As shown in Fig. 2A, all genes studied achieved medium expression stability with M values ranging from 1.09 for B2M to 0.82 for GAPDH (average geNorm M≤1.0), which is typically seen when evaluating candidate reference targets on heterogeneous samples, like cancer biopsies or samples from different tissues. GAPDH, H3F3A, PPIA and HPRT1 were identified as the most stable genes, followed by UBC as the fifth most stable gene. Furthermore, in addition to the generated M stability value, the geNorm program calculates a normalization factor, based on the variable V as the pairwise variation between two sequential normalization, to assess the optimal number of genes required for a reliable normalization of qPCR data. As shown in fig. 2B, taking 0.15 as a cut-off value below which the inclusion of an additional control gene is not required [11], the optimal number of reference targets was 4. As such, based on geNorm analysis, in our experimental situation the optimal normalization factor could be calculated as the geometric mean of reference targets HPRT1, PPIA, H3F3A and GAPDH. Additionally, we compared and validated the results generated from geNorm using the NormFinder program, that selected UBC and PPIA with a stability value of 0.365 and 0.370 respectively as the most stable genes, followed by HPRT1, H3F3A and GAPDH closely behind (Table 4). GeNorm and Normfinder results are in considerably closer agreement in the identification of B2M, ACTB and POLR2A as the least stable genes in our cohort of samples.


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)

Selection of the most suitable reference genes for normalization in endometrial cancer samples using geNorm analysis.Results are presented according to the output file of the geNorm program [11]. (A) Average expression stability value (M) of control genes calculated by a stepwise exclusion of the least stable genes: the program enables elimination of the worst-scoring gene with the highest M value and recalculation of new M value for the remaining genes. X axis from the left to the right indicates gene rank according to expression stability, while Y axis indicate the stability measure M. (B) Determination of the optimal number of control genes for normalization.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113781-g002: Selection of the most suitable reference genes for normalization in endometrial cancer samples using geNorm analysis.Results are presented according to the output file of the geNorm program [11]. (A) Average expression stability value (M) of control genes calculated by a stepwise exclusion of the least stable genes: the program enables elimination of the worst-scoring gene with the highest M value and recalculation of new M value for the remaining genes. X axis from the left to the right indicates gene rank according to expression stability, while Y axis indicate the stability measure M. (B) Determination of the optimal number of control genes for normalization.
Mentions: From the theoretical point of view, genes whose expression are equivalent between malignant and non malignant tissue samples are all suitable reference for relative quantification of target genes in gene profiling studies. Thus, according to the statistical analysis performed on the expression level of the 10 selected reference genes, GUSB, HPRT1 and PPIA were the best candidate for normalization purposes in gene expression studies in endometrial cancer versus normal tissue. To validate and confirm this result, all 10 reference genes including the best performing GUSB, HPRT1 and PPIA were included in the program geNorm which calculated the gene expression stability measure M of one gene based on the average pairwise variation between all studied genes. The lowest M value characterized the gene with the most stable expression. As shown in Fig. 2A, all genes studied achieved medium expression stability with M values ranging from 1.09 for B2M to 0.82 for GAPDH (average geNorm M≤1.0), which is typically seen when evaluating candidate reference targets on heterogeneous samples, like cancer biopsies or samples from different tissues. GAPDH, H3F3A, PPIA and HPRT1 were identified as the most stable genes, followed by UBC as the fifth most stable gene. Furthermore, in addition to the generated M stability value, the geNorm program calculates a normalization factor, based on the variable V as the pairwise variation between two sequential normalization, to assess the optimal number of genes required for a reliable normalization of qPCR data. As shown in fig. 2B, taking 0.15 as a cut-off value below which the inclusion of an additional control gene is not required [11], the optimal number of reference targets was 4. As such, based on geNorm analysis, in our experimental situation the optimal normalization factor could be calculated as the geometric mean of reference targets HPRT1, PPIA, H3F3A and GAPDH. Additionally, we compared and validated the results generated from geNorm using the NormFinder program, that selected UBC and PPIA with a stability value of 0.365 and 0.370 respectively as the most stable genes, followed by HPRT1, H3F3A and GAPDH closely behind (Table 4). GeNorm and Normfinder results are in considerably closer agreement in the identification of B2M, ACTB and POLR2A as the least stable genes in our cohort of samples.

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