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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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Expression level of 32 candidate reference genes in 24 tissues, developmental stages and seedlings exposed to high and low temperatures. The expression levels (Ct values) of each candidate reference gene (UniGene cluster) are shown as average (diagonal cross), median (horizontal lines), 25th to 75th percentile (boxes) and expression ranges (whiskers) for the 24 cDNA pools analysed. The line at Ct 22.31, corresponding to the general mean, discriminates the reference genes with high and low expression.
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Figure 3: Expression level of 32 candidate reference genes in 24 tissues, developmental stages and seedlings exposed to high and low temperatures. The expression levels (Ct values) of each candidate reference gene (UniGene cluster) are shown as average (diagonal cross), median (horizontal lines), 25th to 75th percentile (boxes) and expression ranges (whiskers) for the 24 cDNA pools analysed. The line at Ct 22.31, corresponding to the general mean, discriminates the reference genes with high and low expression.

Mentions: In order to avoid possible errors due to the interaction of co-regulated genes, further analyses were carried out on 32 genes involved in distinct biological processes and metabolic pathways; they comprised 28 genes selected on the basis of their uniform expression detected in UniGene and TIGR and a single gene for each of the four gene families. The expression levels could be grouped into two arbitrary categories: 19 highly expressed genes, whose mean Ct values were below the general mean (Ct = 22.31 cycles) and 13 lowly expressed genes, with mean Ct values above this value (Fig. 3). The highest expression was shown by Ta38797 (Histone), Ta53919 (S-adenosylmethionine decarboxylase), Ta50503 (Ubiquitin) and Ta54825 (Actin), with mean Ct values between 20 and 21 cycles, the lowest by Ta25534 (α-tubulin), Ta2776 (RNase L inhibitor-like protein), Ta54448 and Ta54733 (Proteins of unknown function), and Ta44405 (β-tubulin), with mean Ct values between 26 and 24 cycles (Fig. 3). On the basis of the number of EST sequences included in each Unigene cluster, the 19 reference genes with low Ct values had been previously classified either as highly expressed (9 genes with more than 800 EST) and moderately expressed (10 genes with more than 300 EST). Of the 13 reference genes with low Ct values, only three including more than 300 ESTs (Ta54963, Ta54447 and 54948) were considered moderately expressed in UniGene, whereas the expression level of the remaining 10 genes was low (<300/>100 ESTs) or very low (<100 ESTs). In our opinion, these data indicate a substantial agreement between the gene expression levels evaluated by qRT-PCR analyses and those estimated by the number of ESTs included in each corresponding UniGene cluster. Ta54963 (GABA-receptor associated protein), Ta54238 (Ubiquitin-conjugating enzyme), Ta35497 (Cyclophilin), Ta53967 (Vacuolar ATP synthase 16 kDa proteolipid subunit) exhibited the lowest gene expression variation (less than 3 cycles), while Ta44405 (β-tubulin), Ta38797 (Histone) and Ta1698 (NADP-isocitrate dehydrogenase) showed the most variable expression (more than 5 cycles).


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)

Expression level of 32 candidate reference genes in 24 tissues, developmental stages and seedlings exposed to high and low temperatures. The expression levels (Ct values) of each candidate reference gene (UniGene cluster) are shown as average (diagonal cross), median (horizontal lines), 25th to 75th percentile (boxes) and expression ranges (whiskers) for the 24 cDNA pools analysed. The line at Ct 22.31, corresponding to the general mean, discriminates the reference genes with high and low expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Expression level of 32 candidate reference genes in 24 tissues, developmental stages and seedlings exposed to high and low temperatures. The expression levels (Ct values) of each candidate reference gene (UniGene cluster) are shown as average (diagonal cross), median (horizontal lines), 25th to 75th percentile (boxes) and expression ranges (whiskers) for the 24 cDNA pools analysed. The line at Ct 22.31, corresponding to the general mean, discriminates the reference genes with high and low expression.
Mentions: In order to avoid possible errors due to the interaction of co-regulated genes, further analyses were carried out on 32 genes involved in distinct biological processes and metabolic pathways; they comprised 28 genes selected on the basis of their uniform expression detected in UniGene and TIGR and a single gene for each of the four gene families. The expression levels could be grouped into two arbitrary categories: 19 highly expressed genes, whose mean Ct values were below the general mean (Ct = 22.31 cycles) and 13 lowly expressed genes, with mean Ct values above this value (Fig. 3). The highest expression was shown by Ta38797 (Histone), Ta53919 (S-adenosylmethionine decarboxylase), Ta50503 (Ubiquitin) and Ta54825 (Actin), with mean Ct values between 20 and 21 cycles, the lowest by Ta25534 (α-tubulin), Ta2776 (RNase L inhibitor-like protein), Ta54448 and Ta54733 (Proteins of unknown function), and Ta44405 (β-tubulin), with mean Ct values between 26 and 24 cycles (Fig. 3). On the basis of the number of EST sequences included in each Unigene cluster, the 19 reference genes with low Ct values had been previously classified either as highly expressed (9 genes with more than 800 EST) and moderately expressed (10 genes with more than 300 EST). Of the 13 reference genes with low Ct values, only three including more than 300 ESTs (Ta54963, Ta54447 and 54948) were considered moderately expressed in UniGene, whereas the expression level of the remaining 10 genes was low (<300/>100 ESTs) or very low (<100 ESTs). In our opinion, these data indicate a substantial agreement between the gene expression levels evaluated by qRT-PCR analyses and those estimated by the number of ESTs included in each corresponding UniGene cluster. Ta54963 (GABA-receptor associated protein), Ta54238 (Ubiquitin-conjugating enzyme), Ta35497 (Cyclophilin), Ta53967 (Vacuolar ATP synthase 16 kDa proteolipid subunit) exhibited the lowest gene expression variation (less than 3 cycles), while Ta44405 (β-tubulin), Ta38797 (Histone) and Ta1698 (NADP-isocitrate dehydrogenase) showed the most variable expression (more than 5 cycles).

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