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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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Related in: MedlinePlus

Dissociation curves (A) and agarose gel of PCR products (B) amplified with the primer pairs designed in conserved regions [Actin (m), α-tubulin (m), TEF-1α (m) and ADP-RF (m)] to amplify transcripts of multiple homologous genes for each of the four families.
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Figure 2: Dissociation curves (A) and agarose gel of PCR products (B) amplified with the primer pairs designed in conserved regions [Actin (m), α-tubulin (m), TEF-1α (m) and ADP-RF (m)] to amplify transcripts of multiple homologous genes for each of the four families.

Mentions: In addition to the primer pairs targeting single transcripts, we set out the evaluation of a further subset of primer pairs, designed in conserved regions identified by sequence comparison, to amplify in a single PCR reaction the transcripts of multiple members of some gene families. Primer pairs designed as TEF-1α (m), ADP-RF (m), Actin (m) and α-tubulin (m), where (m) denotes multiple amplifications, targeted conserved sequences within the transcripts of at least two homologous genes for each family. The simultaneous amplification by conserved primer pairs of sequences transcribed from multiple homologous genes with balanced expression levels has been proposed as a suitable strategy for qRT-PCR normalization. Such an approach has been exploited for the Arabidopsis genes AtACT2 and AtACT8, which display complementary patterns of expression, making their combined expression quasi-constitutive [52]. Gel electrophoresis and melting-curve analysis showed that each of the four conserved primer pairs designed to amplify transcripts from multiple genes of the four analysed families of wheat amplified a PCR product of the expected size from the various cDNA pools (Fig. 2). Single PCR products purified from gels were cloned into the pGEM plasmid vector (Promega) and ten different clones were sequenced for each of the four multiple amplifications. Two sequences of 276 bp and with identity of 90.58% were observed among the ten clones obtained by the PCR with the primer pair ADP-RF (m); also among the ten clones produced by the amplification using the primer pair TEF-1α (m) there were two different sequences of 233 bp with 95.28% identity. In both cases the sequences corresponded to those used for designing the primer pairs within the conserved regions, Ta45379/Ta2291 for ADP-RF and Ta659/Ta53964 for TEF-1α. In each group of ten clones obtained by the primer pairs Actin (m) and α-tubulin (m) three similar homologous sequences were found, which matched three of the five members of their respective gene families (Ta54825/Ta53908/Ta20863 for Actin and Ta53981/Ta33558/Ta54519 for α-tubulin), showing that multiple amplifications were produced also by these conserved primers.


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)

Dissociation curves (A) and agarose gel of PCR products (B) amplified with the primer pairs designed in conserved regions [Actin (m), α-tubulin (m), TEF-1α (m) and ADP-RF (m)] to amplify transcripts of multiple homologous genes for each of the four families.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Dissociation curves (A) and agarose gel of PCR products (B) amplified with the primer pairs designed in conserved regions [Actin (m), α-tubulin (m), TEF-1α (m) and ADP-RF (m)] to amplify transcripts of multiple homologous genes for each of the four families.
Mentions: In addition to the primer pairs targeting single transcripts, we set out the evaluation of a further subset of primer pairs, designed in conserved regions identified by sequence comparison, to amplify in a single PCR reaction the transcripts of multiple members of some gene families. Primer pairs designed as TEF-1α (m), ADP-RF (m), Actin (m) and α-tubulin (m), where (m) denotes multiple amplifications, targeted conserved sequences within the transcripts of at least two homologous genes for each family. The simultaneous amplification by conserved primer pairs of sequences transcribed from multiple homologous genes with balanced expression levels has been proposed as a suitable strategy for qRT-PCR normalization. Such an approach has been exploited for the Arabidopsis genes AtACT2 and AtACT8, which display complementary patterns of expression, making their combined expression quasi-constitutive [52]. Gel electrophoresis and melting-curve analysis showed that each of the four conserved primer pairs designed to amplify transcripts from multiple genes of the four analysed families of wheat amplified a PCR product of the expected size from the various cDNA pools (Fig. 2). Single PCR products purified from gels were cloned into the pGEM plasmid vector (Promega) and ten different clones were sequenced for each of the four multiple amplifications. Two sequences of 276 bp and with identity of 90.58% were observed among the ten clones obtained by the PCR with the primer pair ADP-RF (m); also among the ten clones produced by the amplification using the primer pair TEF-1α (m) there were two different sequences of 233 bp with 95.28% identity. In both cases the sequences corresponded to those used for designing the primer pairs within the conserved regions, Ta45379/Ta2291 for ADP-RF and Ta659/Ta53964 for TEF-1α. In each group of ten clones obtained by the primer pairs Actin (m) and α-tubulin (m) three similar homologous sequences were found, which matched three of the five members of their respective gene families (Ta54825/Ta53908/Ta20863 for Actin and Ta53981/Ta33558/Ta54519 for α-tubulin), showing that multiple amplifications were produced also by these conserved primers.

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