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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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Specificity of qRT-PCR amplification. (A) Dissociation curves of six representative reference genes showing single peaks (each including three technical replicates for each of 18 cDNA pools from different tissues and developmental stages of wheat). (B) Agarose gel (2%) showing amplification of a specific PCR product of the expected size for the same genes in (A). Dissociation curves and agarose gels for all the 42 candidate reference genes selected are shown in Additional file 8.
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Figure 1: Specificity of qRT-PCR amplification. (A) Dissociation curves of six representative reference genes showing single peaks (each including three technical replicates for each of 18 cDNA pools from different tissues and developmental stages of wheat). (B) Agarose gel (2%) showing amplification of a specific PCR product of the expected size for the same genes in (A). Dissociation curves and agarose gels for all the 42 candidate reference genes selected are shown in Additional file 8.

Mentions: Since we chose a two-step qRT-PCR protocol, reverse transcription and PCR-mediated cDNA amplification were carried out by subsequent steps in separate tubes. The two-step protocol was preferred because it would reduce unwanted dimer formation between primers when SYBR Green is used as a detection dye [51]. The RT reaction was primed by oligo-(dT), rather than using random-sequence primers which anneal preferentially to abundant mRNA species. This choice was critical because, as deduced on the basis of the number of EST sequences comprised in the corresponding UniGene cluster, some of the selected candidate reference genes would be expressed at low level. PCR primer pairs for the 32 selected reference candidate genes and for the five members for each of the α-tubulin and actin gene families were designed and preliminarily tested in qRT-PCR reactions from a pool of all available cDNAs (see Methods). In order to avoid unreliable amplification of splice variants and assure uniform RT efficiencies, typically primer pairs were designed to target an amplicon located within the 3' end region of the transcript of interest. For each biological replicate and tissue sample, the same cDNA pool was used for qRT-PCR amplification of the 42 (32+10) analysed genes using the previously tested gene-specific primers. Moreover, qRT-PCR analysis of every gene was performed in triplicate for each of the 24 cDNA pools, along with no template and RT-minus controls. The 42 primer pairs used to amplify the candidate reference genes generated single amplicons of the expected size from the various cDNA pools, as shown by the presence of single bands in agarose gel electrophoresis and by single-peak melting curves of the PCR products obtained after 40 amplification cycles (Fig. 1 and Additional file 8). A detailed analysis of the dissociation curves confirmed the absence of primer dimers and of other products resulting from non-specific amplification (Additional file 8). A more stringent test of PCR specificity was performed by sequencing the amplification products of each of the 42 candidate reference genes. Always the sequence of the PCR product matched that of the target cDNA, thereby confirming the exquisite PCR specificity of the developed primer pairs.


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)

Specificity of qRT-PCR amplification. (A) Dissociation curves of six representative reference genes showing single peaks (each including three technical replicates for each of 18 cDNA pools from different tissues and developmental stages of wheat). (B) Agarose gel (2%) showing amplification of a specific PCR product of the expected size for the same genes in (A). Dissociation curves and agarose gels for all the 42 candidate reference genes selected are shown in Additional file 8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Specificity of qRT-PCR amplification. (A) Dissociation curves of six representative reference genes showing single peaks (each including three technical replicates for each of 18 cDNA pools from different tissues and developmental stages of wheat). (B) Agarose gel (2%) showing amplification of a specific PCR product of the expected size for the same genes in (A). Dissociation curves and agarose gels for all the 42 candidate reference genes selected are shown in Additional file 8.
Mentions: Since we chose a two-step qRT-PCR protocol, reverse transcription and PCR-mediated cDNA amplification were carried out by subsequent steps in separate tubes. The two-step protocol was preferred because it would reduce unwanted dimer formation between primers when SYBR Green is used as a detection dye [51]. The RT reaction was primed by oligo-(dT), rather than using random-sequence primers which anneal preferentially to abundant mRNA species. This choice was critical because, as deduced on the basis of the number of EST sequences comprised in the corresponding UniGene cluster, some of the selected candidate reference genes would be expressed at low level. PCR primer pairs for the 32 selected reference candidate genes and for the five members for each of the α-tubulin and actin gene families were designed and preliminarily tested in qRT-PCR reactions from a pool of all available cDNAs (see Methods). In order to avoid unreliable amplification of splice variants and assure uniform RT efficiencies, typically primer pairs were designed to target an amplicon located within the 3' end region of the transcript of interest. For each biological replicate and tissue sample, the same cDNA pool was used for qRT-PCR amplification of the 42 (32+10) analysed genes using the previously tested gene-specific primers. Moreover, qRT-PCR analysis of every gene was performed in triplicate for each of the 24 cDNA pools, along with no template and RT-minus controls. The 42 primer pairs used to amplify the candidate reference genes generated single amplicons of the expected size from the various cDNA pools, as shown by the presence of single bands in agarose gel electrophoresis and by single-peak melting curves of the PCR products obtained after 40 amplification cycles (Fig. 1 and Additional file 8). A detailed analysis of the dissociation curves confirmed the absence of primer dimers and of other products resulting from non-specific amplification (Additional file 8). A more stringent test of PCR specificity was performed by sequencing the amplification products of each of the 42 candidate reference genes. Always the sequence of the PCR product matched that of the target cDNA, thereby confirming the exquisite PCR specificity of the developed primer pairs.

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