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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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Coefficient of variation calculation for the potential reference genes. This bar chart displays the coefficient of variation (CV) in % of the 20 potential reference genes across the gravity conditions for the 19th DLR PFC (H/W, 1 g, 1.8 g, μg) and TEXUS-49 (H/W, BL, μg). A lower value corresponds to higher stability in gene expression. (a) 19th DLR PFC: All calculated CV values are below the threshold of 50%. (b) TEXUS-49: all CV values are below 50%, but in total more genes show higher coefficients of variation.
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fig3: Coefficient of variation calculation for the potential reference genes. This bar chart displays the coefficient of variation (CV) in % of the 20 potential reference genes across the gravity conditions for the 19th DLR PFC (H/W, 1 g, 1.8 g, μg) and TEXUS-49 (H/W, BL, μg). A lower value corresponds to higher stability in gene expression. (a) 19th DLR PFC: All calculated CV values are below the threshold of 50%. (b) TEXUS-49: all CV values are below 50%, but in total more genes show higher coefficients of variation.

Mentions: In search of potential reference genes for gravitational studies in this monocytic/macrophageal cell system, we first performed PubMed database search to identify commonly used reference genes in RNA expression analyses in human cells. We found 22 genes that were used in several reverse transcription quantitative real-time PCR (RT-qPCR) studies as control genes for normalization (Table 2, supplementary Table 2). The microarray data tables were screened for these 22 widely used reference genes, and 20 of them could be located on the Roche NimbleGen 12 × 135 K array that was used in our experiments. Two genes coding for 5s and 18s rRNAs could not be identified, since they are not spotted on the array. The PFC and TEXUS data sets were screened for those 20 selected potential reference genes, and fluorescence intensities were compiled for each gene and each gravity condition in heatmaps (Figures 2(a) and 2(b)). Overall fluorescence intensities for all samples showed only minor differences in the heatmaps. A more detailed visual inspection revealed completely equal fluorescence intensities for ACTB, ALB, B4GALT6, HMBS, HPRT1, PPIA, RPLP0, and YWHAZ for the gravity conditions prevailing during the 19th DLR PFC (Figure 2(a)). The gravity conditions investigated during the TEXUS-49 campaign showed stable expression values for the genes ACTB, ALB, B4GALT6, GUSB, PLA2G4A, POLR2A, PPIA, TBP, UBC, and YWHAZ (Figure 2(b)). For further characterization and identification of stable reference genes, we performed a geNorm pilot study [51] and calculated the coefficient of variation (CV) for all 20 potential reference genes (Figure 3). For homogeneous groups, CV values below 25%, and for heterogeneous groups, CV values below 50% are acceptable [56]. Rapid and extreme changes in gravity induce strong changes in cellular functions. Therefore, we classified our samples as heterogeneous groups. According to the set criteria, all analyzed potential reference genes showed CV values below 50% for the PFC and TEXUS data sets (Figures 3(a) and 3(b)). In the sample set of the 19th DLR PFC, all genes but HMBS fulfill even the more stringent criterion of a CV below 25% (Figure 3(a)). For the samples collected during the TEXUS-49 campaign, all genes but HPRT1 and PLA2G4A display CV values below 25% (Figure 3(b)).


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)

Coefficient of variation calculation for the potential reference genes. This bar chart displays the coefficient of variation (CV) in % of the 20 potential reference genes across the gravity conditions for the 19th DLR PFC (H/W, 1 g, 1.8 g, μg) and TEXUS-49 (H/W, BL, μg). A lower value corresponds to higher stability in gene expression. (a) 19th DLR PFC: All calculated CV values are below the threshold of 50%. (b) TEXUS-49: all CV values are below 50%, but in total more genes show higher coefficients of variation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Coefficient of variation calculation for the potential reference genes. This bar chart displays the coefficient of variation (CV) in % of the 20 potential reference genes across the gravity conditions for the 19th DLR PFC (H/W, 1 g, 1.8 g, μg) and TEXUS-49 (H/W, BL, μg). A lower value corresponds to higher stability in gene expression. (a) 19th DLR PFC: All calculated CV values are below the threshold of 50%. (b) TEXUS-49: all CV values are below 50%, but in total more genes show higher coefficients of variation.
Mentions: In search of potential reference genes for gravitational studies in this monocytic/macrophageal cell system, we first performed PubMed database search to identify commonly used reference genes in RNA expression analyses in human cells. We found 22 genes that were used in several reverse transcription quantitative real-time PCR (RT-qPCR) studies as control genes for normalization (Table 2, supplementary Table 2). The microarray data tables were screened for these 22 widely used reference genes, and 20 of them could be located on the Roche NimbleGen 12 × 135 K array that was used in our experiments. Two genes coding for 5s and 18s rRNAs could not be identified, since they are not spotted on the array. The PFC and TEXUS data sets were screened for those 20 selected potential reference genes, and fluorescence intensities were compiled for each gene and each gravity condition in heatmaps (Figures 2(a) and 2(b)). Overall fluorescence intensities for all samples showed only minor differences in the heatmaps. A more detailed visual inspection revealed completely equal fluorescence intensities for ACTB, ALB, B4GALT6, HMBS, HPRT1, PPIA, RPLP0, and YWHAZ for the gravity conditions prevailing during the 19th DLR PFC (Figure 2(a)). The gravity conditions investigated during the TEXUS-49 campaign showed stable expression values for the genes ACTB, ALB, B4GALT6, GUSB, PLA2G4A, POLR2A, PPIA, TBP, UBC, and YWHAZ (Figure 2(b)). For further characterization and identification of stable reference genes, we performed a geNorm pilot study [51] and calculated the coefficient of variation (CV) for all 20 potential reference genes (Figure 3). For homogeneous groups, CV values below 25%, and for heterogeneous groups, CV values below 50% are acceptable [56]. Rapid and extreme changes in gravity induce strong changes in cellular functions. Therefore, we classified our samples as heterogeneous groups. According to the set criteria, all analyzed potential reference genes showed CV values below 50% for the PFC and TEXUS data sets (Figures 3(a) and 3(b)). In the sample set of the 19th DLR PFC, all genes but HMBS fulfill even the more stringent criterion of a CV below 25% (Figure 3(a)). For the samples collected during the TEXUS-49 campaign, all genes but HPRT1 and PLA2G4A display CV values below 25% (Figure 3(b)).

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