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Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

Thiel CS, Hauschild S, Tauber S, Paulsen K, Raig C, Raem A, Biskup J, Gutewort A, Hürlimann E, Unverdorben F, Buttron I, Lauber B, Philpot C, Lier H, Engelmann F, Layer LE, Ullrich O - Biomed Res Int (2015)

Bottom Line: However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet.In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems.We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, Faculty of Medicine, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland ; Department of Machine Design, Engineering Design and Product Development, Institute of Mechanical Engineering, Otto-von-Guericke-University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany ; Study Group "Magdeburger Arbeitsgemeinschaft für Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen" (MARS), Otto-von-Guericke-University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany.

ABSTRACT
Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

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Heatmaps for highly conserved ABC transporters. The fluorescent intensity levels of the 47 ABC transporter genes shown in supplementary Table 2 were quantified for the different gravity conditions. Each gene is represented in one column, and each gravity condition is represented in one row. (a) μg, 1 g, H/W, and 1.8 g (19th DLR PFC) and (b) μg, BL, and H/W (TEXUS-49). The heatmaps show large variation in fluorescence intensities for the different genes. However, within the same gene, expression levels are mostly similar for all tested conditions. The lower bar with the graduated red colors is the measure for the different fluorescence intensities.
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fig4: Heatmaps for highly conserved ABC transporters. The fluorescent intensity levels of the 47 ABC transporter genes shown in supplementary Table 2 were quantified for the different gravity conditions. Each gene is represented in one column, and each gravity condition is represented in one row. (a) μg, 1 g, H/W, and 1.8 g (19th DLR PFC) and (b) μg, BL, and H/W (TEXUS-49). The heatmaps show large variation in fluorescence intensities for the different genes. However, within the same gene, expression levels are mostly similar for all tested conditions. The lower bar with the graduated red colors is the measure for the different fluorescence intensities.

Mentions: To increase the number of potential reference genes that can be used as standards for differential expression analyses in gravitational studies, we extended our analysis to evolutionary highly conserved genes. We hypothesized that genes stable over time and taxonomic kingdoms should have very fundamental functions within a cell and thus be largely independent from external influences to ensure basic cellular functions. Besides ribosomal RNA genes, which are not represented on the microarray applied in this study, ABC transporters and tRNA genes are also evolutionary highly conserved over a wide variety of organisms. Unfortunately, the 12 × 135 K Roche NimbleGen array does also not contain probes for tRNA. Therefore, we had a look at expression profiles of ABC transporters and tRNA related genes (supplementary Tables 2 and 3). Since almost all fluorescence values of tRNA related genes showed a high variance making reasonable analysis impossible, we concentrated on the ABC transporters. Heatmap analyses were carried out to obtain a first impression on the gene stability (Figure 4). The samples from the 19th DLR PFC and TEXUS-49 mission also show a rather high variation in fluorescence intensities (Figures 4(a) and 4(b)). The calculation of the CV for these samples (Figure 5) displays higher values compared to the potential reference genes; however, taken together all analyzed samples of the 19th DLR PFC fulfill the criterion of CV values less than 50% in case of ABC transporter signals (Figure 5(a)). Out of 47 samples, 36 are also below 25% CV. Although three samples from TEXUS-49 showed values above 50% CV (Figure 5(b)), 37 samples stayed below the 25% threshold (Figure 5(b)). Selected reference genes and ABC transporters (marked in bold, Figures 4 and 5) were chosen for further detailed analysis of differential gene expression under altered gravity conditions.


Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

Thiel CS, Hauschild S, Tauber S, Paulsen K, Raig C, Raem A, Biskup J, Gutewort A, Hürlimann E, Unverdorben F, Buttron I, Lauber B, Philpot C, Lier H, Engelmann F, Layer LE, Ullrich O - Biomed Res Int (2015)

Heatmaps for highly conserved ABC transporters. The fluorescent intensity levels of the 47 ABC transporter genes shown in supplementary Table 2 were quantified for the different gravity conditions. Each gene is represented in one column, and each gravity condition is represented in one row. (a) μg, 1 g, H/W, and 1.8 g (19th DLR PFC) and (b) μg, BL, and H/W (TEXUS-49). The heatmaps show large variation in fluorescence intensities for the different genes. However, within the same gene, expression levels are mostly similar for all tested conditions. The lower bar with the graduated red colors is the measure for the different fluorescence intensities.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309215&req=5

fig4: Heatmaps for highly conserved ABC transporters. The fluorescent intensity levels of the 47 ABC transporter genes shown in supplementary Table 2 were quantified for the different gravity conditions. Each gene is represented in one column, and each gravity condition is represented in one row. (a) μg, 1 g, H/W, and 1.8 g (19th DLR PFC) and (b) μg, BL, and H/W (TEXUS-49). The heatmaps show large variation in fluorescence intensities for the different genes. However, within the same gene, expression levels are mostly similar for all tested conditions. The lower bar with the graduated red colors is the measure for the different fluorescence intensities.
Mentions: To increase the number of potential reference genes that can be used as standards for differential expression analyses in gravitational studies, we extended our analysis to evolutionary highly conserved genes. We hypothesized that genes stable over time and taxonomic kingdoms should have very fundamental functions within a cell and thus be largely independent from external influences to ensure basic cellular functions. Besides ribosomal RNA genes, which are not represented on the microarray applied in this study, ABC transporters and tRNA genes are also evolutionary highly conserved over a wide variety of organisms. Unfortunately, the 12 × 135 K Roche NimbleGen array does also not contain probes for tRNA. Therefore, we had a look at expression profiles of ABC transporters and tRNA related genes (supplementary Tables 2 and 3). Since almost all fluorescence values of tRNA related genes showed a high variance making reasonable analysis impossible, we concentrated on the ABC transporters. Heatmap analyses were carried out to obtain a first impression on the gene stability (Figure 4). The samples from the 19th DLR PFC and TEXUS-49 mission also show a rather high variation in fluorescence intensities (Figures 4(a) and 4(b)). The calculation of the CV for these samples (Figure 5) displays higher values compared to the potential reference genes; however, taken together all analyzed samples of the 19th DLR PFC fulfill the criterion of CV values less than 50% in case of ABC transporter signals (Figure 5(a)). Out of 47 samples, 36 are also below 25% CV. Although three samples from TEXUS-49 showed values above 50% CV (Figure 5(b)), 37 samples stayed below the 25% threshold (Figure 5(b)). Selected reference genes and ABC transporters (marked in bold, Figures 4 and 5) were chosen for further detailed analysis of differential gene expression under altered gravity conditions.

Bottom Line: However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet.In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems.We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, Faculty of Medicine, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland ; Department of Machine Design, Engineering Design and Product Development, Institute of Mechanical Engineering, Otto-von-Guericke-University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany ; Study Group "Magdeburger Arbeitsgemeinschaft für Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen" (MARS), Otto-von-Guericke-University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany.

ABSTRACT
Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

Show MeSH