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Identification and validation of reference genes for quantitative RT-PCR normalization in wheat.

Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M - BMC Mol. Biol. (2009)

Bottom Line: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use.Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat.The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy. Annarita0711@libero.it

ABSTRACT

Background: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR.

Results: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and alpha-tubulin.

Conclusion: The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

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Relative quantification of TaPDIL1-1 transcripts in three data sets using selected reference genes as internal controls. A) Six vegetative tissues and shoot developmental stages (shoots, stems and leaves); B) six floral organs from fully emerged spikes; C) six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls (LT = low temperature; HT = high temperature). The TaPDIL1-1 expression levels were normalized using the five methods described in Fig. 5. Normalized values of TaPDIL1-1 relative expression are given as average ± SD.
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Figure 6: Relative quantification of TaPDIL1-1 transcripts in three data sets using selected reference genes as internal controls. A) Six vegetative tissues and shoot developmental stages (shoots, stems and leaves); B) six floral organs from fully emerged spikes; C) six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls (LT = low temperature; HT = high temperature). The TaPDIL1-1 expression levels were normalized using the five methods described in Fig. 5. Normalized values of TaPDIL1-1 relative expression are given as average ± SD.

Mentions: Normalization of the expression of TaPDIL-1-1 using the reference genes selected by the three methods based on geNorm and NormFinder programmes did not detect significant differences in the relative expression of the gene of interest in all the four analysed data sets (Figs. 5 and 6). As expected, the mRNA of TaPDIL1-1 was constitutively expressed in all analysed tissues, with very high transcription at the early stage of seed development (Seeds-1) and moderately high expression also in the middle and late stages of spike development (Spikes 2–3) and in floral organs such as lodicules and pistil. The lowest transcription was detected in vegetative tissues, such as mature leaves and stems, and in the non-reproductive floral organs (glumes, lemma and palea) (Fig. 5). On the contrary, normalization of the TaPDIL1-1 transcription levels by the commonly used reference genes Ta54825 (Actin) and Ta25534 (α-tubulin) led to significant over- or under-estimation of transcription of the gene of interest (Fig. 5). Normalization using Ta25534 (α-tubulin) of the TaPDIL-1 expression in spikes collected at different stages of development, for instance, led to an estimation of its transcription level about 2–6 times higher than those obtained using the most stable genes indicated by geNorm and NormFinder (Fig. 5). The misleading effect of using unsuitable reference genes for the normalization of expression data was even more evident when the analysis was performed on the data sets relative to single groups of tissues (vegetative or floral organs, Figs. 6A and 6B) or temperature treatments (Fig. 6C). In the data set including only the vegetative tissues normalization with Ta54825 (Actin) estimated a significantly lower expression level of TaPDIL1-1 (approximately 1.3–3.6 times) in four of the six tissues analysed (Shoots1, Shoots3, Shoots4 and leaves) (Fig. 6A). Moreover, the normalization procedure with Ta25534 (α-tubulin) showed a weak but significant enhancement of TaPDIL1-1 mRNA expression in shoots collected from plants at three leaves unfolded stage, at the beginning of tillering and with formed tillers (Shoots1-3 in Fig. 6A). Among the floral organs, the use of Ta54825 (Actin) and Ta25534 (α-tubulin) as single reference genes led to significant over-estimation of TaPDIL1-1 expression in lodicules and pistil (Fig. 6B). Moreover, when TaPDIL1-1 expression was normalized with Ta54825 (Actin) in stamens its transcription level was about 3.7 times higher than the estimation obtained using the best genes indicated by geNorm and NormFinder (Fig. 6B). Finally, when the normalization of the TaPDIL1-1 gene was carried out using the geometric mean of the reference genes selected by NormFinder and by geNorm, the single best gene identified by NormFinder and Ta54825 (Actin), the comparison of seedlings exposed for 48 h at low (4°C) and high (33°C) temperatures with the control samples exposed to 18°C detected a significant up-regulation of TaPDIL1-1 mRNA (Fig. 6C). It is noteworthy that Ta54825 (Actin) was one of the two most stable genes selected by geNorm in the temperature treatments (Table 3). On the contrary, the normalization of TaPDIL1-1 expression data using Ta25534 (α-tubulin) as reference gene resulted in a significant decrement of its transcription level after 24 and 48 h exposure to low and high temperatures (Fig. 6C). These contrasting results can be explained by the significant up-regulation of Ta25534 (α-tubulin) induced by the cold and heat shocks.


Identification and validation of reference genes for quantitative RT-PCR normalization in wheat.

Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M - BMC Mol. Biol. (2009)

Relative quantification of TaPDIL1-1 transcripts in three data sets using selected reference genes as internal controls. A) Six vegetative tissues and shoot developmental stages (shoots, stems and leaves); B) six floral organs from fully emerged spikes; C) six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls (LT = low temperature; HT = high temperature). The TaPDIL1-1 expression levels were normalized using the five methods described in Fig. 5. Normalized values of TaPDIL1-1 relative expression are given as average ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667184&req=5

Figure 6: Relative quantification of TaPDIL1-1 transcripts in three data sets using selected reference genes as internal controls. A) Six vegetative tissues and shoot developmental stages (shoots, stems and leaves); B) six floral organs from fully emerged spikes; C) six samples consisting of two temperature treatments (4°C and 33°C) for 24 and 48 h and their controls (LT = low temperature; HT = high temperature). The TaPDIL1-1 expression levels were normalized using the five methods described in Fig. 5. Normalized values of TaPDIL1-1 relative expression are given as average ± SD.
Mentions: Normalization of the expression of TaPDIL-1-1 using the reference genes selected by the three methods based on geNorm and NormFinder programmes did not detect significant differences in the relative expression of the gene of interest in all the four analysed data sets (Figs. 5 and 6). As expected, the mRNA of TaPDIL1-1 was constitutively expressed in all analysed tissues, with very high transcription at the early stage of seed development (Seeds-1) and moderately high expression also in the middle and late stages of spike development (Spikes 2–3) and in floral organs such as lodicules and pistil. The lowest transcription was detected in vegetative tissues, such as mature leaves and stems, and in the non-reproductive floral organs (glumes, lemma and palea) (Fig. 5). On the contrary, normalization of the TaPDIL1-1 transcription levels by the commonly used reference genes Ta54825 (Actin) and Ta25534 (α-tubulin) led to significant over- or under-estimation of transcription of the gene of interest (Fig. 5). Normalization using Ta25534 (α-tubulin) of the TaPDIL-1 expression in spikes collected at different stages of development, for instance, led to an estimation of its transcription level about 2–6 times higher than those obtained using the most stable genes indicated by geNorm and NormFinder (Fig. 5). The misleading effect of using unsuitable reference genes for the normalization of expression data was even more evident when the analysis was performed on the data sets relative to single groups of tissues (vegetative or floral organs, Figs. 6A and 6B) or temperature treatments (Fig. 6C). In the data set including only the vegetative tissues normalization with Ta54825 (Actin) estimated a significantly lower expression level of TaPDIL1-1 (approximately 1.3–3.6 times) in four of the six tissues analysed (Shoots1, Shoots3, Shoots4 and leaves) (Fig. 6A). Moreover, the normalization procedure with Ta25534 (α-tubulin) showed a weak but significant enhancement of TaPDIL1-1 mRNA expression in shoots collected from plants at three leaves unfolded stage, at the beginning of tillering and with formed tillers (Shoots1-3 in Fig. 6A). Among the floral organs, the use of Ta54825 (Actin) and Ta25534 (α-tubulin) as single reference genes led to significant over-estimation of TaPDIL1-1 expression in lodicules and pistil (Fig. 6B). Moreover, when TaPDIL1-1 expression was normalized with Ta54825 (Actin) in stamens its transcription level was about 3.7 times higher than the estimation obtained using the best genes indicated by geNorm and NormFinder (Fig. 6B). Finally, when the normalization of the TaPDIL1-1 gene was carried out using the geometric mean of the reference genes selected by NormFinder and by geNorm, the single best gene identified by NormFinder and Ta54825 (Actin), the comparison of seedlings exposed for 48 h at low (4°C) and high (33°C) temperatures with the control samples exposed to 18°C detected a significant up-regulation of TaPDIL1-1 mRNA (Fig. 6C). It is noteworthy that Ta54825 (Actin) was one of the two most stable genes selected by geNorm in the temperature treatments (Table 3). On the contrary, the normalization of TaPDIL1-1 expression data using Ta25534 (α-tubulin) as reference gene resulted in a significant decrement of its transcription level after 24 and 48 h exposure to low and high temperatures (Fig. 6C). These contrasting results can be explained by the significant up-regulation of Ta25534 (α-tubulin) induced by the cold and heat shocks.

Bottom Line: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use.Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat.The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy. Annarita0711@libero.it

ABSTRACT

Background: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR.

Results: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and alpha-tubulin.

Conclusion: The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.

Show MeSH
Related in: MedlinePlus