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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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Boxplots showing the log expression values of individual microarrays. The central line represents the 50th percentile or median, whereas the upper and lower boundaries of the box display the 75th and 25th percentile, respectively. The upper and lower bars represent the 9th and the 91st percentile. Two experimental data sets are displayed, (a) 28 microarrays hybridized with samples from the 19th DLR PFC (8x μg, 6x H/W, 8x 1 g, 6x 1.8 g) and (b) 18 microarrays hybridized with samples originating from the TEXUS-49 campaign (7x μg, 6x H/W, 5x 1 g). The expression data show an even distribution for the displayed log intensities.
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fig1: Boxplots showing the log expression values of individual microarrays. The central line represents the 50th percentile or median, whereas the upper and lower boundaries of the box display the 75th and 25th percentile, respectively. The upper and lower bars represent the 9th and the 91st percentile. Two experimental data sets are displayed, (a) 28 microarrays hybridized with samples from the 19th DLR PFC (8x μg, 6x H/W, 8x 1 g, 6x 1.8 g) and (b) 18 microarrays hybridized with samples originating from the TEXUS-49 campaign (7x μg, 6x H/W, 5x 1 g). The expression data show an even distribution for the displayed log intensities.

Mentions: Data tables were compiled individually for the 19th DLR PFC and TEXUS-49 including all gravity conditions listed in Table 1, and a first overview of the datasets was provided by a boxplot diagram (Figures 1(a) and 1(b)). Boxplots are a useful tool to visualize the variation within a microarray and between microarrays. The central line shows the position of the median, while the upper and the lower boundaries represent the upper (75th percentile) and lower (25th percentile) quartile. The ends of the tails display the 9th and the 91st percentile. The boxplots of the microarray data show that there is only little variation within a single array and between the arrays that belong to the same gravity condition. Figure 1 shows that the quality of both data sets (19th DLR PFC and TEXUS-49) is sufficient to proceed with further analyses.


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)

Boxplots showing the log expression values of individual microarrays. The central line represents the 50th percentile or median, whereas the upper and lower boundaries of the box display the 75th and 25th percentile, respectively. The upper and lower bars represent the 9th and the 91st percentile. Two experimental data sets are displayed, (a) 28 microarrays hybridized with samples from the 19th DLR PFC (8x μg, 6x H/W, 8x 1 g, 6x 1.8 g) and (b) 18 microarrays hybridized with samples originating from the TEXUS-49 campaign (7x μg, 6x H/W, 5x 1 g). The expression data show an even distribution for the displayed log intensities.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Boxplots showing the log expression values of individual microarrays. The central line represents the 50th percentile or median, whereas the upper and lower boundaries of the box display the 75th and 25th percentile, respectively. The upper and lower bars represent the 9th and the 91st percentile. Two experimental data sets are displayed, (a) 28 microarrays hybridized with samples from the 19th DLR PFC (8x μg, 6x H/W, 8x 1 g, 6x 1.8 g) and (b) 18 microarrays hybridized with samples originating from the TEXUS-49 campaign (7x μg, 6x H/W, 5x 1 g). The expression data show an even distribution for the displayed log intensities.
Mentions: Data tables were compiled individually for the 19th DLR PFC and TEXUS-49 including all gravity conditions listed in Table 1, and a first overview of the datasets was provided by a boxplot diagram (Figures 1(a) and 1(b)). Boxplots are a useful tool to visualize the variation within a microarray and between microarrays. The central line shows the position of the median, while the upper and the lower boundaries represent the upper (75th percentile) and lower (25th percentile) quartile. The ends of the tails display the 9th and the 91st percentile. The boxplots of the microarray data show that there is only little variation within a single array and between the arrays that belong to the same gravity condition. Figure 1 shows that the quality of both data sets (19th DLR PFC and TEXUS-49) is sufficient to proceed with further analyses.

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