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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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Boxplots showing the Ct variation of the reference genes. The variation in Ct values of the eight candidate reference genes in (A) the CA samples (3 biological replicates for each time point, n = 24) and in (B) the ovary (3 biological replicates for each time point, n = 24) as indicated by the raw Ct values. The values are given as the cycle threshold (Ct, mean of triplicate samples). The boxplots show the 25th quartile, median and the 75th quartile (horizontal lines) and the minimal and maximal values (whiskers).
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Figure 2: Boxplots showing the Ct variation of the reference genes. The variation in Ct values of the eight candidate reference genes in (A) the CA samples (3 biological replicates for each time point, n = 24) and in (B) the ovary (3 biological replicates for each time point, n = 24) as indicated by the raw Ct values. The values are given as the cycle threshold (Ct, mean of triplicate samples). The boxplots show the 25th quartile, median and the 75th quartile (horizontal lines) and the minimal and maximal values (whiskers).

Mentions: Variation of the Ct values among the 24 samples for the eight candidate reference genes is shown in Figure 2. Raw Ct values are given in Additional file 2. In the CA, the Ct values for the selected reference genes ranged from 20.33 (EF1a) to 30.62 (Arm). In the ovary, the Ct values ranged from 18.65 (EF1a) to 28.23 (Arm). Transcripts for EF1a and Arm were shown to be the most and least abundant expressions respectively in both tissue types tested. The Ct values between CA and ovary can, however, not be compared since the amount of RNA used to transcribe to cDNA was not the same.


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)

Boxplots showing the Ct variation of the reference genes. The variation in Ct values of the eight candidate reference genes in (A) the CA samples (3 biological replicates for each time point, n = 24) and in (B) the ovary (3 biological replicates for each time point, n = 24) as indicated by the raw Ct values. The values are given as the cycle threshold (Ct, mean of triplicate samples). The boxplots show the 25th quartile, median and the 75th quartile (horizontal lines) and the minimal and maximal values (whiskers).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Boxplots showing the Ct variation of the reference genes. The variation in Ct values of the eight candidate reference genes in (A) the CA samples (3 biological replicates for each time point, n = 24) and in (B) the ovary (3 biological replicates for each time point, n = 24) as indicated by the raw Ct values. The values are given as the cycle threshold (Ct, mean of triplicate samples). The boxplots show the 25th quartile, median and the 75th quartile (horizontal lines) and the minimal and maximal values (whiskers).
Mentions: Variation of the Ct values among the 24 samples for the eight candidate reference genes is shown in Figure 2. Raw Ct values are given in Additional file 2. In the CA, the Ct values for the selected reference genes ranged from 20.33 (EF1a) to 30.62 (Arm). In the ovary, the Ct values ranged from 18.65 (EF1a) to 28.23 (Arm). Transcripts for EF1a and Arm were shown to be the most and least abundant expressions respectively in both tissue types tested. The Ct values between CA and ovary can, however, not be compared since the amount of RNA used to transcribe to cDNA was not the same.

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