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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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Influence of altered gravity during parabolic flight on potential reference genes. RNA expression levels after 1 g (light gray), 1.8 g (dark gray), and μg (black) conditions during the 19th DLR parabolic flight campaign. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA expression levels are shown as fluorescence intensities. (a) The expression values for GAPDH, HMBS, RPLP0, TBP, and YWHAZ are displayed. (b) ALB and B4GALT6 show low but stable fluorescent intensities. GAPDH, HMBS, YWHAZ, ALB, and B4GALT6 show no significant change in RNA levels upon altered gravity for 20 sec, while RPLP0 displays μg sensitivity compared to 1 g and TBP reacts sensitively to all g conditions. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005.
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fig6: Influence of altered gravity during parabolic flight on potential reference genes. RNA expression levels after 1 g (light gray), 1.8 g (dark gray), and μg (black) conditions during the 19th DLR parabolic flight campaign. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA expression levels are shown as fluorescence intensities. (a) The expression values for GAPDH, HMBS, RPLP0, TBP, and YWHAZ are displayed. (b) ALB and B4GALT6 show low but stable fluorescent intensities. GAPDH, HMBS, YWHAZ, ALB, and B4GALT6 show no significant change in RNA levels upon altered gravity for 20 sec, while RPLP0 displays μg sensitivity compared to 1 g and TBP reacts sensitively to all g conditions. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005.

Mentions: For nine of the potential reference genes from the literature, there were at least three values returned by the microarray, generated by independent probes targeting the same gene. Two of these genes were excluded from further analysis due to high variance between their single values (HSP90AA1 and PPIA), and the remaining seven genes (ALB, B4GALT6, GAPDH, HMBS, RPLP0, TBP, and YWHAZ (see Table 3)) were subjected to further statistics. The calculation of the mean fluorescence intensity levels revealed that different ranges of transcript abundance are present in both experimental setups. While ALB and B4GALT6 seem to be expressed rather low, GAPDH, HMBS, RPLP0, TBP, and YWHAZ are represented in much higher abundance (Figures 6 and 7). The comparison of mean fluorescence intensities of one gene under different g conditions revealed that GAPDH, HMBS, YWHAZ, ALB, and B4GALT6 are stably expressed with respect to all investigated gravity conditions during parabolic flight of the 19th DLR PFC (Figures 6(a) and 6(b)). RPLP0 is significantly upregulated by μg compared to 1 g, while TBP is initially downregulated by 1.8 g and recovers during μg (Figure 6(a)). Furthermore, comparison of in-flight 1 g controls to 1 g ground controls (H/W) shows a significantly reduced mRNA level of TBP portending that during the preexperimental phase a certain kind of stress was accumulated in the cells influencing its expression level.


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)

Influence of altered gravity during parabolic flight on potential reference genes. RNA expression levels after 1 g (light gray), 1.8 g (dark gray), and μg (black) conditions during the 19th DLR parabolic flight campaign. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA expression levels are shown as fluorescence intensities. (a) The expression values for GAPDH, HMBS, RPLP0, TBP, and YWHAZ are displayed. (b) ALB and B4GALT6 show low but stable fluorescent intensities. GAPDH, HMBS, YWHAZ, ALB, and B4GALT6 show no significant change in RNA levels upon altered gravity for 20 sec, while RPLP0 displays μg sensitivity compared to 1 g and TBP reacts sensitively to all g conditions. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Influence of altered gravity during parabolic flight on potential reference genes. RNA expression levels after 1 g (light gray), 1.8 g (dark gray), and μg (black) conditions during the 19th DLR parabolic flight campaign. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA expression levels are shown as fluorescence intensities. (a) The expression values for GAPDH, HMBS, RPLP0, TBP, and YWHAZ are displayed. (b) ALB and B4GALT6 show low but stable fluorescent intensities. GAPDH, HMBS, YWHAZ, ALB, and B4GALT6 show no significant change in RNA levels upon altered gravity for 20 sec, while RPLP0 displays μg sensitivity compared to 1 g and TBP reacts sensitively to all g conditions. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005.
Mentions: For nine of the potential reference genes from the literature, there were at least three values returned by the microarray, generated by independent probes targeting the same gene. Two of these genes were excluded from further analysis due to high variance between their single values (HSP90AA1 and PPIA), and the remaining seven genes (ALB, B4GALT6, GAPDH, HMBS, RPLP0, TBP, and YWHAZ (see Table 3)) were subjected to further statistics. The calculation of the mean fluorescence intensity levels revealed that different ranges of transcript abundance are present in both experimental setups. While ALB and B4GALT6 seem to be expressed rather low, GAPDH, HMBS, RPLP0, TBP, and YWHAZ are represented in much higher abundance (Figures 6 and 7). The comparison of mean fluorescence intensities of one gene under different g conditions revealed that GAPDH, HMBS, YWHAZ, ALB, and B4GALT6 are stably expressed with respect to all investigated gravity conditions during parabolic flight of the 19th DLR PFC (Figures 6(a) and 6(b)). RPLP0 is significantly upregulated by μg compared to 1 g, while TBP is initially downregulated by 1.8 g and recovers during μg (Figure 6(a)). Furthermore, comparison of in-flight 1 g controls to 1 g ground controls (H/W) shows a significantly reduced mRNA level of TBP portending that during the preexperimental phase a certain kind of stress was accumulated in the cells influencing its expression level.

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