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Identification of most stable endogenous control genes for microRNA quantification in the developing mouse lung.

Bouhaddioui W, Provost PR, Tremblay Y - PLoS ONE (2014)

Bottom Line: Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0.In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development.The use of normalization genes selected for their expression stability is essential in lung development studies.

View Article: PubMed Central - PubMed

Affiliation: Reproduction, Mother and Youth Health, Centre de recherche CHU de Québec, Québec, QC, Canada; Centre de Recherche en Biologie de la Reproduction (CRBR), Faculté de Médecine, Université Laval, Québec, QC, Canada.

ABSTRACT
MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies.

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Relative expression levels of Let-7a in saccular-stage lungs normalized with different pairs of housekeeping genes.Let7-a RNA levels obtained by qPCR are presented for lung RNA samples collected during the saccular stage on GD 18.0 and PN 0. Data were normalized using the best pair of control genes calculated with the saccular stage samples and with: A) geNorm (sno135/sno142); B) NormFinder (sno142/sno251). In (C), a pair of less stable genes as estimated either by geNorm or NormFinder was used (sno202/sno234) and a significant sex difference was observed on PN 0 (*, P = 0.018, Student t-test). When the best pair calculated by geNorm using all the samples from pseudoglandular to alveolar stages (sno135/sno234) was used (D), no significant sex difference was observed on PN 0 (P = 0.109, Student t-test). Pools of male and female lungs were used (see Table 1 for details).
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pone-0111855-g004: Relative expression levels of Let-7a in saccular-stage lungs normalized with different pairs of housekeeping genes.Let7-a RNA levels obtained by qPCR are presented for lung RNA samples collected during the saccular stage on GD 18.0 and PN 0. Data were normalized using the best pair of control genes calculated with the saccular stage samples and with: A) geNorm (sno135/sno142); B) NormFinder (sno142/sno251). In (C), a pair of less stable genes as estimated either by geNorm or NormFinder was used (sno202/sno234) and a significant sex difference was observed on PN 0 (*, P = 0.018, Student t-test). When the best pair calculated by geNorm using all the samples from pseudoglandular to alveolar stages (sno135/sno234) was used (D), no significant sex difference was observed on PN 0 (P = 0.109, Student t-test). Pools of male and female lungs were used (see Table 1 for details).

Mentions: To determine the effect of the selection of normalization genes on quantification of miRNA expression, we quantified Let-7a RNA levels using different pairs of control genes. First, we selected the best pairs of genes sorted by geNorm and NormFinder for the canalicular stage and compared the results with those obtained with another pair of genes. The magnitude of the sex difference in let7-a expression levels evaluated with the two proposed pairs of genes was similar (3.8× vs 4.8×), whereas the data normalized with the least stable pair of genes showed higher sex differences (10.6×) (Fig. 3). Second, we reproduced the same experiment but using samples from GD 18.0 and PN 0 of the saccular stage separately. No statistically significant sex difference in let7-a expression levels was observed on GD 18.0 with either the best pairs of normalization genes calculated with all the samples of the saccular stage by geNorm (sno135/sno142) and NormFinder (sno142/sno251), or the least stable control genes (sno234/sno202) (Fig. 4). However, for males on GD 18.0, a higher variability between biological replicates was observed with the best control genes selected by geNorm compared with those selected by NormFinder. For an unknown reason, such a higher variability was not observed for all the experimental conditions normalized with this pair of control genes (sno135/sno142). With samples from PN 0, no sex difference was observed in let7-a expression levels using the best pair of control genes from the two calculation methods, while a statistically significant sex difference (p<0.02) was observed with the other pair. Therefore, the use of the two calculation methods led to similar conclusions, in contrast to the use of the less stable pair of normalization genes. We also used the most stable pair of genes calculated by geNorm with data from the entire studied developmental window (sno135/sno234). When normalized with these control genes, the let7-a expression data showed no statistically significant sex difference, which is the same conclusion as with the best stable control genes calculated with geNorm and NormFinder using only the samples from the saccular stage (Fig. 4D). The fact that the developing lung is changing implies that normalization genes should be selected within the analyzed time window. The use of control genes selected from multi-stage sampling would be reserved for studies extending over multiple developmental stages. Taken together, our data demonstrate the importance of choosing the most stable pair of endogenous control genes to adequately represent the actual biological situation.


Identification of most stable endogenous control genes for microRNA quantification in the developing mouse lung.

Bouhaddioui W, Provost PR, Tremblay Y - PLoS ONE (2014)

Relative expression levels of Let-7a in saccular-stage lungs normalized with different pairs of housekeeping genes.Let7-a RNA levels obtained by qPCR are presented for lung RNA samples collected during the saccular stage on GD 18.0 and PN 0. Data were normalized using the best pair of control genes calculated with the saccular stage samples and with: A) geNorm (sno135/sno142); B) NormFinder (sno142/sno251). In (C), a pair of less stable genes as estimated either by geNorm or NormFinder was used (sno202/sno234) and a significant sex difference was observed on PN 0 (*, P = 0.018, Student t-test). When the best pair calculated by geNorm using all the samples from pseudoglandular to alveolar stages (sno135/sno234) was used (D), no significant sex difference was observed on PN 0 (P = 0.109, Student t-test). Pools of male and female lungs were used (see Table 1 for details).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219792&req=5

pone-0111855-g004: Relative expression levels of Let-7a in saccular-stage lungs normalized with different pairs of housekeeping genes.Let7-a RNA levels obtained by qPCR are presented for lung RNA samples collected during the saccular stage on GD 18.0 and PN 0. Data were normalized using the best pair of control genes calculated with the saccular stage samples and with: A) geNorm (sno135/sno142); B) NormFinder (sno142/sno251). In (C), a pair of less stable genes as estimated either by geNorm or NormFinder was used (sno202/sno234) and a significant sex difference was observed on PN 0 (*, P = 0.018, Student t-test). When the best pair calculated by geNorm using all the samples from pseudoglandular to alveolar stages (sno135/sno234) was used (D), no significant sex difference was observed on PN 0 (P = 0.109, Student t-test). Pools of male and female lungs were used (see Table 1 for details).
Mentions: To determine the effect of the selection of normalization genes on quantification of miRNA expression, we quantified Let-7a RNA levels using different pairs of control genes. First, we selected the best pairs of genes sorted by geNorm and NormFinder for the canalicular stage and compared the results with those obtained with another pair of genes. The magnitude of the sex difference in let7-a expression levels evaluated with the two proposed pairs of genes was similar (3.8× vs 4.8×), whereas the data normalized with the least stable pair of genes showed higher sex differences (10.6×) (Fig. 3). Second, we reproduced the same experiment but using samples from GD 18.0 and PN 0 of the saccular stage separately. No statistically significant sex difference in let7-a expression levels was observed on GD 18.0 with either the best pairs of normalization genes calculated with all the samples of the saccular stage by geNorm (sno135/sno142) and NormFinder (sno142/sno251), or the least stable control genes (sno234/sno202) (Fig. 4). However, for males on GD 18.0, a higher variability between biological replicates was observed with the best control genes selected by geNorm compared with those selected by NormFinder. For an unknown reason, such a higher variability was not observed for all the experimental conditions normalized with this pair of control genes (sno135/sno142). With samples from PN 0, no sex difference was observed in let7-a expression levels using the best pair of control genes from the two calculation methods, while a statistically significant sex difference (p<0.02) was observed with the other pair. Therefore, the use of the two calculation methods led to similar conclusions, in contrast to the use of the less stable pair of normalization genes. We also used the most stable pair of genes calculated by geNorm with data from the entire studied developmental window (sno135/sno234). When normalized with these control genes, the let7-a expression data showed no statistically significant sex difference, which is the same conclusion as with the best stable control genes calculated with geNorm and NormFinder using only the samples from the saccular stage (Fig. 4D). The fact that the developing lung is changing implies that normalization genes should be selected within the analyzed time window. The use of control genes selected from multi-stage sampling would be reserved for studies extending over multiple developmental stages. Taken together, our data demonstrate the importance of choosing the most stable pair of endogenous control genes to adequately represent the actual biological situation.

Bottom Line: Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0.In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development.The use of normalization genes selected for their expression stability is essential in lung development studies.

View Article: PubMed Central - PubMed

Affiliation: Reproduction, Mother and Youth Health, Centre de recherche CHU de Québec, Québec, QC, Canada; Centre de Recherche en Biologie de la Reproduction (CRBR), Faculté de Médecine, Université Laval, Québec, QC, Canada.

ABSTRACT
MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies.

Show MeSH
Related in: MedlinePlus