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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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JH and oocyte measurements of the test animals. The right-hand axis shows the length of basal oocytes dissected from D. punctata females during the first gonadotrophic cycle (day 0 to day 6 after the imaginal molt, oviposition took place at the start of day 7). The data represent means of 30 individual animals. Vertical bars indicate standard deviations. The left-hand axis represents the amount of JH released during the first gonadotrophic cycle (day 0 to day 7 after final molt). The data are represented in pmol/hour per individual CA and represent means of 12 independent biological replicates. The vertical bars indicate standard errors.
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Figure 1: JH and oocyte measurements of the test animals. The right-hand axis shows the length of basal oocytes dissected from D. punctata females during the first gonadotrophic cycle (day 0 to day 6 after the imaginal molt, oviposition took place at the start of day 7). The data represent means of 30 individual animals. Vertical bars indicate standard deviations. The left-hand axis represents the amount of JH released during the first gonadotrophic cycle (day 0 to day 7 after final molt). The data are represented in pmol/hour per individual CA and represent means of 12 independent biological replicates. The vertical bars indicate standard errors.

Mentions: Oocyte length was determined for each dissected female cockroach used in the q-RT-PCR assays (Figure 1). A clear rising trend in length can be observed as vitellogenin is taken into the developing oocytes, as was previously described [28]. The sample animals ovulated at the start of day 7. Rates of JH release throughout the gonadotrophic cycle steadily rise until four days post final molt, a sharp decline can be seen on day 5 (Figure 1). The activity of the CA is linked to the cycle of egg development, as was reported by Tobe and Stay [21]: Rates of JH release increase as oocytes grow during vitellogenesis. Before oviposition, JH release decreases and remains at a low level during gestation.


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)

JH and oocyte measurements of the test animals. The right-hand axis shows the length of basal oocytes dissected from D. punctata females during the first gonadotrophic cycle (day 0 to day 6 after the imaginal molt, oviposition took place at the start of day 7). The data represent means of 30 individual animals. Vertical bars indicate standard deviations. The left-hand axis represents the amount of JH released during the first gonadotrophic cycle (day 0 to day 7 after final molt). The data are represented in pmol/hour per individual CA and represent means of 12 independent biological replicates. The vertical bars indicate standard errors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: JH and oocyte measurements of the test animals. The right-hand axis shows the length of basal oocytes dissected from D. punctata females during the first gonadotrophic cycle (day 0 to day 6 after the imaginal molt, oviposition took place at the start of day 7). The data represent means of 30 individual animals. Vertical bars indicate standard deviations. The left-hand axis represents the amount of JH released during the first gonadotrophic cycle (day 0 to day 7 after final molt). The data are represented in pmol/hour per individual CA and represent means of 12 independent biological replicates. The vertical bars indicate standard errors.
Mentions: Oocyte length was determined for each dissected female cockroach used in the q-RT-PCR assays (Figure 1). A clear rising trend in length can be observed as vitellogenin is taken into the developing oocytes, as was previously described [28]. The sample animals ovulated at the start of day 7. Rates of JH release throughout the gonadotrophic cycle steadily rise until four days post final molt, a sharp decline can be seen on day 5 (Figure 1). The activity of the CA is linked to the cycle of egg development, as was reported by Tobe and Stay [21]: Rates of JH release increase as oocytes grow during vitellogenesis. Before oviposition, JH release decreases and remains at a low level during gestation.

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
Related in: MedlinePlus