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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

Leaves, stems and roots used for RNA extraction. Mature leaves, stem epidermis and healthy roots were collected from salt-stressed P. euphratica saplings. Bars = 2 cm.
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ijms-16-20468-f001: Leaves, stems and roots used for RNA extraction. Mature leaves, stem epidermis and healthy roots were collected from salt-stressed P. euphratica saplings. Bars = 2 cm.

Mentions: This study was performed in accordance with the minimum information for publication of reverse transcription-quantitative PCR experiments (MIQE)-guidelines (Table S1). P. euphratica saplings (Figure S1) were exposed to salt stress for 0, 1, 3, 6, 9 and 12 h and then leaves, stems and roots were sampled for RNA extraction (Figure 1). The time-point “0” was used as a reference point, and sampling at further time-points represents a combination of salt-induced changes in gene expression as well as diurnal changes. Based on agarose gel electrophoresis, the intensity of the 25S rRNA band was nearly twice that of 18S rRNA band, and no genomic DNA was observed (Figure S2). Using a spectrophotometer, the OD260/OD280 ratios of total RNA were between 1.8 and 2.0, and the OD260/OD230 ratios were greater than 1.5. Using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) for total RNA integrity detection, no evidence of degradation was observed. After cDNA synthesis, qPCR using the primers listed in Table 1 was performed, and to verify the specificity of these primers, the amplified products were analyzed using 2% agarose gel electrophoresis and only one band of the expected size was observed in each experiment. Meanwhile, also the presence of a single peak in the melting curve was observed (Figure S3).


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)

Leaves, stems and roots used for RNA extraction. Mature leaves, stem epidermis and healthy roots were collected from salt-stressed P. euphratica saplings. Bars = 2 cm.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-20468-f001: Leaves, stems and roots used for RNA extraction. Mature leaves, stem epidermis and healthy roots were collected from salt-stressed P. euphratica saplings. Bars = 2 cm.
Mentions: This study was performed in accordance with the minimum information for publication of reverse transcription-quantitative PCR experiments (MIQE)-guidelines (Table S1). P. euphratica saplings (Figure S1) were exposed to salt stress for 0, 1, 3, 6, 9 and 12 h and then leaves, stems and roots were sampled for RNA extraction (Figure 1). The time-point “0” was used as a reference point, and sampling at further time-points represents a combination of salt-induced changes in gene expression as well as diurnal changes. Based on agarose gel electrophoresis, the intensity of the 25S rRNA band was nearly twice that of 18S rRNA band, and no genomic DNA was observed (Figure S2). Using a spectrophotometer, the OD260/OD280 ratios of total RNA were between 1.8 and 2.0, and the OD260/OD230 ratios were greater than 1.5. Using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) for total RNA integrity detection, no evidence of degradation was observed. After cDNA synthesis, qPCR using the primers listed in Table 1 was performed, and to verify the specificity of these primers, the amplified products were analyzed using 2% agarose gel electrophoresis and only one band of the expected size was observed in each experiment. Meanwhile, also the presence of a single peak in the melting curve was observed (Figure S3).

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