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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.

Show MeSH
Influence of altered gravity during sounding rocket flight on ABC transporter genes. ABCC1, ABCC4, ABCD4, ABCF2, and TAP2 RNA expression levels after launch and acceleration (BL, dark gray) and μg (black) conditions of TEXUS-49. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA levels are depicted as fluorescence intensities. Only ABCC1 expression is stable over all g conditions. ABCC4, ABCD4, ABCF2, and TAP2 display μg sensitivity compared to BL and to H/W in the case of ABCF2 and TAP2. TAP2 also shows vibration sensitivity comparing BL to H/W. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005, and ***P < 0.0005.
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fig9: Influence of altered gravity during sounding rocket flight on ABC transporter genes. ABCC1, ABCC4, ABCD4, ABCF2, and TAP2 RNA expression levels after launch and acceleration (BL, dark gray) and μg (black) conditions of TEXUS-49. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA levels are depicted as fluorescence intensities. Only ABCC1 expression is stable over all g conditions. ABCC4, ABCD4, ABCF2, and TAP2 display μg sensitivity compared to BL and to H/W in the case of ABCF2 and TAP2. TAP2 also shows vibration sensitivity comparing BL to H/W. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005, and ***P < 0.0005.

Mentions: The highly conserved ABC transporters were represented as a large group of genes on the applied microarray. We analyzed a total of 47 ABC transporters belonging to nine different sub-families (supplementary Table 2). 19 ABC transporter genes were represented by three or more individual probes on the microarray and 11 of them had similar fluorescent intensities meeting the requirements for a statistical analysis. Exemplarily, five of those 11 ABC transporters are shown in Figures 8 and 9.


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 sounding rocket flight on ABC transporter genes. ABCC1, ABCC4, ABCD4, ABCF2, and TAP2 RNA expression levels after launch and acceleration (BL, dark gray) and μg (black) conditions of TEXUS-49. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA levels are depicted as fluorescence intensities. Only ABCC1 expression is stable over all g conditions. ABCC4, ABCD4, ABCF2, and TAP2 display μg sensitivity compared to BL and to H/W in the case of ABCF2 and TAP2. TAP2 also shows vibration sensitivity comparing BL to H/W. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005, and ***P < 0.0005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Influence of altered gravity during sounding rocket flight on ABC transporter genes. ABCC1, ABCC4, ABCD4, ABCF2, and TAP2 RNA expression levels after launch and acceleration (BL, dark gray) and μg (black) conditions of TEXUS-49. Hardware ground controls (H/W, striped) are shown for each experimental group. RNA levels are depicted as fluorescence intensities. Only ABCC1 expression is stable over all g conditions. ABCC4, ABCD4, ABCF2, and TAP2 display μg sensitivity compared to BL and to H/W in the case of ABCF2 and TAP2. TAP2 also shows vibration sensitivity comparing BL to H/W. Mean values of at least three measurements with standard deviations are shown. *P < 0.05, **P < 0.005, and ***P < 0.0005.
Mentions: The highly conserved ABC transporters were represented as a large group of genes on the applied microarray. We analyzed a total of 47 ABC transporters belonging to nine different sub-families (supplementary Table 2). 19 ABC transporter genes were represented by three or more individual probes on the microarray and 11 of them had similar fluorescent intensities meeting the requirements for a statistical analysis. Exemplarily, five of those 11 ABC transporters are shown in Figures 8 and 9.

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