Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

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.

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4

“…Table 3 summarizes those genes, which were previously identified to be stably expressed over all analyzed gravity conditions from seconds to minutes and which we suggested as suitable reference genes for platform spanning microgravity research of cells of the monocyte–macrophage system. 22 We performed the reference gene analysis for the Jurkat T cell expression data set with all the genes listed in Tables 2 and 3 . The mean values and standard deviations were calculated from all single expression data from all samples for each gene and platform and the coefficient of variation value was determined (Fig.…”

“…Within the scope of this investigation and by comparing the results to former studies in monocytic cells, 22 we also aimed at identifying reliable and comprehensive reference genes for differential gene expression analysis performed with immune cells on the major Earth-based platforms providing real and simulated microgravity. Reference genes (“housekeeping genes”) are essential to verify and standardize gene expression analysis as internal controls, also for the amount and quality of starting material and the reaction efficiency.…”

“…Therefore, we also re-evaluated 20 reference genes identified earlier as widely applied throughout the literature 22 using microarray data for these particular genes in human Jurkat T lymphocytes 23 and assessed their stability. 23 , 24 Finally, our study aims to estimate to which extent changes of the gravity environment influence gene expression homeostasis compared to other environmental stress factors and to provide therefore new data for an appropriate risk assessment of long-term space missions.…”

SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogenLeishmania donovani

“…Total RNA of replica samples used for immunoblot analysis was extracted, and reverse transcription (RT) qPCR was performed for selected transcripts. The transcripts of 60S ribosomal protein L19 (RpL19/P84099) and 14-3-3 protein zeta/delta (Ywhaz/P63101) were used as normalisation controls, as these reference genes are considered to have stable expression [47, 48] in these culture conditions. Certain changes in transcript levels correlated with changes in protein abundance, including reduced abundance for Pppt1 and increased abundance for PlxnA1 and the copper-transporting ATPase 1 (Atp7/Q64430).…”

Schnelle zelluläre Reaktion und Anpassung an Schwerelosigkeit

“…In unseren Studien untersuchten wir Signaltransduktionskaskaden [3,[6][7][8], Adhäsionsmoleküle [9][10][11], Stoffwechsel [11], funktionelle Parameter [12,13], Genexpressionsreaktionen [6,[14][15][16][17][18][19]…”