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Sequencing and validation of housekeeping genes for quantitative real-time PCR during the gonadotrophic cycle of Diploptera punctata.

Marchal E, Hult EF, Huang J, Tobe SS - BMC Res Notes (2013)

Bottom Line: Accurate and reliable results depend on the use of stable reference genes for normalization.Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata.Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Canada.

ABSTRACT

Background: Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression.

Results: Candidate housekeeping genes were sequenced: β-actin (Actin), elongation factor 1 alpha (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), armadillo (Arm), ribosomal protein L32 (RpL32), succinate dehydrogenase (SDHa), annexin IX (AnnIX) and α-tubulin (Tub). The expression of these eight genes was analyzed in corpora allata (CA) and ovaries of adult female D. punctata. Both geNorm, as well as Normfinder characterized SDHa, EF1a and Arm as being the most stably expressed in the corpora allata. In the ovary, the geNorm calculation showed Tub, EF1a and RpL32 to be most stable, whereas Normfinder identified Tub, EF1a and Arm as the best. In ovary, the least stable gene was Actin, challenging its usefulness in normalization. As a proof of principle, the expression of follicle cell protein 3c and CYP15A1 was monitored during the first gonadotrophic cycle.

Conclusion: Arm and EF1a form the most stably expressed combination of two reference genes out of the eight candidates that were tested in the corpora allata. Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata. Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata. The results stress the necessity for validation of reference genes in q-RT-PCR studies in cockroaches.

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Optimal number of reference genes for normalization as calculated by geNorm. Pairwise variation analysis determining the optimal number of reference genes required ensuring accurate normalization between normalization factors NFn and NFn+1 in (A) CA and (B) ovary during the gonadotrophic cycle.
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Figure 5: Optimal number of reference genes for normalization as calculated by geNorm. Pairwise variation analysis determining the optimal number of reference genes required ensuring accurate normalization between normalization factors NFn and NFn+1 in (A) CA and (B) ovary during the gonadotrophic cycle.

Mentions: In addition, geNorm can also determine the optimal number of genes required for accurate normalization. Upon inclusion of a less stable reference gene, geNorm calculates the pairwise variation (V) using sequential normalization factors (NFn and NFn + 1). The algorithm can thus determine if addition of the extra reference gene will add to the stability of the normalization factor. A cut-off value of V = 0.15 is recommended in deciding the addition of the next reference gene. In the CA, the lowest V-value was calculated following inclusion of the 7th most stable gene, this means that addition of the least stable gene AnnIX would negatively impact the normalization process. This is similar in the ovary, in which inclusion of Actin would result in a poorer normalization. Our results indicate that in CA, only two reference genes are necessary for accurate normalization since the V2/3-value was found to be below the cut-off of 0.15 (Figure 5A). In the ovary however, addition of the third most stable gene (EF1a) is needed, but adding a fourth is not imperative (Figure 5B).


Sequencing and validation of housekeeping genes for quantitative real-time PCR during the gonadotrophic cycle of Diploptera punctata.

Marchal E, Hult EF, Huang J, Tobe SS - BMC Res Notes (2013)

Optimal number of reference genes for normalization as calculated by geNorm. Pairwise variation analysis determining the optimal number of reference genes required ensuring accurate normalization between normalization factors NFn and NFn+1 in (A) CA and (B) ovary during the gonadotrophic cycle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750588&req=5

Figure 5: Optimal number of reference genes for normalization as calculated by geNorm. Pairwise variation analysis determining the optimal number of reference genes required ensuring accurate normalization between normalization factors NFn and NFn+1 in (A) CA and (B) ovary during the gonadotrophic cycle.
Mentions: In addition, geNorm can also determine the optimal number of genes required for accurate normalization. Upon inclusion of a less stable reference gene, geNorm calculates the pairwise variation (V) using sequential normalization factors (NFn and NFn + 1). The algorithm can thus determine if addition of the extra reference gene will add to the stability of the normalization factor. A cut-off value of V = 0.15 is recommended in deciding the addition of the next reference gene. In the CA, the lowest V-value was calculated following inclusion of the 7th most stable gene, this means that addition of the least stable gene AnnIX would negatively impact the normalization process. This is similar in the ovary, in which inclusion of Actin would result in a poorer normalization. Our results indicate that in CA, only two reference genes are necessary for accurate normalization since the V2/3-value was found to be below the cut-off of 0.15 (Figure 5A). In the ovary however, addition of the third most stable gene (EF1a) is needed, but adding a fourth is not imperative (Figure 5B).

Bottom Line: Accurate and reliable results depend on the use of stable reference genes for normalization.Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata.Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Canada.

ABSTRACT

Background: Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression.

Results: Candidate housekeeping genes were sequenced: β-actin (Actin), elongation factor 1 alpha (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), armadillo (Arm), ribosomal protein L32 (RpL32), succinate dehydrogenase (SDHa), annexin IX (AnnIX) and α-tubulin (Tub). The expression of these eight genes was analyzed in corpora allata (CA) and ovaries of adult female D. punctata. Both geNorm, as well as Normfinder characterized SDHa, EF1a and Arm as being the most stably expressed in the corpora allata. In the ovary, the geNorm calculation showed Tub, EF1a and RpL32 to be most stable, whereas Normfinder identified Tub, EF1a and Arm as the best. In ovary, the least stable gene was Actin, challenging its usefulness in normalization. As a proof of principle, the expression of follicle cell protein 3c and CYP15A1 was monitored during the first gonadotrophic cycle.

Conclusion: Arm and EF1a form the most stably expressed combination of two reference genes out of the eight candidates that were tested in the corpora allata. Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata. Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata. The results stress the necessity for validation of reference genes in q-RT-PCR studies in cockroaches.

Show MeSH