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Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms.

Wang HL, Li L, Tang S, Yuan C, Tian Q, Su Y, Li HG, Zhao L, Yin W, Zhao R, Xia X - Int J Mol Sci (2015)

Bottom Line: To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues.Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene.These results are valuable for research of gene identification in different P. euphratica tissues.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. whling@bjfu.edu.cn.

ABSTRACT
Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

No MeSH data available.


Related in: MedlinePlus

Rank aggregation of ten genes lists using the Monte Carlo algorithm. Visual representation of rank aggregation using RankAggreg with the Monte Carlo algorithm and Spearman footrule distances. The ten candidate reference genes were ordered based on their rank position according to three stability detection methods, ΔCt, NormFinder and geNorm (gray lines). The mean rank position of each gene is shown in black, while the model computed using the Monte Carlo algorithm is indicated by a red line.
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ijms-16-20468-f005: Rank aggregation of ten genes lists using the Monte Carlo algorithm. Visual representation of rank aggregation using RankAggreg with the Monte Carlo algorithm and Spearman footrule distances. The ten candidate reference genes were ordered based on their rank position according to three stability detection methods, ΔCt, NormFinder and geNorm (gray lines). The mean rank position of each gene is shown in black, while the model computed using the Monte Carlo algorithm is indicated by a red line.

Mentions: Since the different methods showed different rankings results for each gene, we used the RankAggreg statistical method to create an aggregate order to obtain a final list of genes for each tissue. The rank positions generated using the three statistical approaches were merged, including mSD values of ΔCt, stability values of NormFinder, and M values of geNorm. The results in Figure 5 indicate that the most adequate genes tested for normalization in leaf were RP, HIS, and eIF-5A. In stem, the best three candidate genes were UBQ, eIF-5A, and HIS. In addition, eIF-5A, GAPDH and UBQ were top-ranked in root.


Evaluation of Appropriate Reference Genes for Reverse Transcription-Quantitative PCR Studies in Different Tissues of a Desert Poplar via Comparision of Different Algorithms.

Wang HL, Li L, Tang S, Yuan C, Tian Q, Su Y, Li HG, Zhao L, Yin W, Zhao R, Xia X - Int J Mol Sci (2015)

Rank aggregation of ten genes lists using the Monte Carlo algorithm. Visual representation of rank aggregation using RankAggreg with the Monte Carlo algorithm and Spearman footrule distances. The ten candidate reference genes were ordered based on their rank position according to three stability detection methods, ΔCt, NormFinder and geNorm (gray lines). The mean rank position of each gene is shown in black, while the model computed using the Monte Carlo algorithm is indicated by a red line.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4613214&req=5

ijms-16-20468-f005: Rank aggregation of ten genes lists using the Monte Carlo algorithm. Visual representation of rank aggregation using RankAggreg with the Monte Carlo algorithm and Spearman footrule distances. The ten candidate reference genes were ordered based on their rank position according to three stability detection methods, ΔCt, NormFinder and geNorm (gray lines). The mean rank position of each gene is shown in black, while the model computed using the Monte Carlo algorithm is indicated by a red line.
Mentions: Since the different methods showed different rankings results for each gene, we used the RankAggreg statistical method to create an aggregate order to obtain a final list of genes for each tissue. The rank positions generated using the three statistical approaches were merged, including mSD values of ΔCt, stability values of NormFinder, and M values of geNorm. The results in Figure 5 indicate that the most adequate genes tested for normalization in leaf were RP, HIS, and eIF-5A. In stem, the best three candidate genes were UBQ, eIF-5A, and HIS. In addition, eIF-5A, GAPDH and UBQ were top-ranked in root.

Bottom Line: To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues.Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene.These results are valuable for research of gene identification in different P. euphratica tissues.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China. whling@bjfu.edu.cn.

ABSTRACT
Despite the unshakable status of reverse transcription-quantitative PCR in gene expression analysis, it has certain disadvantages, including that the results are highly dependent on the reference genes selected for data normalization. Since inappropriate endogenous control genes will lead to inaccurate target gene expression profiles, the validation of suitable internal reference genes is essential. Given the increasing interest in functional genes and genomics of Populus euphratica, a desert poplar showing extraordinary adaptation to salt stress, we evaluated the expression stability of ten candidate reference genes in P. euphratica roots, stems, and leaves under salt stress conditions. We used five algorithms, namely, ΔCt, NormFinder, geNorm, GrayNorm, and a rank aggregation method (RankAggreg) to identify suitable normalizers. To support the suitability of the identified reference genes and to compare the relative merits of these different algorithms, we analyzed and compared the relative expression levels of nine P. euphratica functional genes in different tissues. Our results indicate that a combination of multiple reference genes recommended by GrayNorm algorithm (e.g., a combination of Actin, EF1α, GAPDH, RP, UBQ in root) should be used instead of a single reference gene. These results are valuable for research of gene identification in different P. euphratica tissues.

No MeSH data available.


Related in: MedlinePlus