Unknown,Transcriptomics,Genomics,Proteomics

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Small Perturbation Approach Reveals Transcriptomic Steady State

ABSTRACT: The â??small perturbationâ?? approach is critical in studying the â??steady stateâ?? of a biological system. In our experiments, small perturbations were generated by applying a series of repeating intermittent small doses of ultraviolet radiation to a human keratinocyte cell line, HaCaT. The biological response was assessed by monitoring the gene expression profiles using a high reliability and high resolution cDNA microarray system. Following intermittent 10 J/m2 UVB small perturbations, two opposite classes of genes, down-regulated and up-regulated, exhibited an immediate response followed by relaxation between each small perturbation, but were prolonged down- or up-regulated without relaxation while larger doses (233 or 582.5 J/m2) of UVB were applied. A repeated cycle pattern of gene expression following small perturbations is an indication of the existence of steady states. This cycle pattern is suppressed when large perturbations are applied. We believe that this is a universal phenomenon. In our experiments, the functions of up-regulated genes were mainly associated with anti-proliferation, anti-mitogenesis, and apoptosis. On the other hand, down-regulated genes were mainly related to proliferation, mitogenesis, and anti-apoptosis. In conclusion, this study experimentally proves the concept of steady state at the transcription level and demonstrates the feasibility of using small perturbation approaches for investigating steady states. This study could also set a foundation of computational systems biology, which has implicitly used the concept of steady state. Keywords: time course Three UVB exposures are indicated by UV at time points of 0, 8 and 16 hours. T1, T2, T3, T4, T5, and T6 denote the sampling time points. T1, T3 and T5 are allocated 30 minutes after the corresponding UVB irradiation. T2, T4 and T6 are allocated 8 hours after each UVB irradiation. At each sampling time point, two samples (control and UV-irradiated) are collected. See supplementary file Loop_design.pdf for further explanation.

ORGANISM(S): Homo sapiens

SUBMITTER: Ian Hsu

PROVIDER: E-GEOD-7060 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ultraviolet (UV) wavebands in sunlight are immunomodulatory. About half the amount of UVA within a minimum erythemal dose of sunlight is systemically immunosuppressive, while higher doses protect from UVB immunosuppression in mice. We have previously shown that these responses to UVA are genetically restricted as they occur in C57BL/6 but not Balb/c mice. We used gene set enrichment analysis of microarray data and real-time RT-PCR confirmation to determine the molecular mechanisms associated with UVA immunomodulation. We found up-regulation of mRNA for the alternative complement pathway. The core-enriched genes complement component 3, properdin and complement factor B were all activated by the immunosuppressive dose of UVA only in UVA-responsive C57BL/6 but not unresponsive BALB/c mice. This therefore matched the genetic restriction and dose responsiveness of UVA immunosuppression. The immune-protective higher UVA dose prevented UVB from down regulating chemokine receptor 7 and IL-12B, and decreased IL-10, supporting previous identification of IL-12 and IL-10 in high dose UVA protection from UVB immunosuppression. Our study has identified activation of the alternative complement pathway as a trigger of UVA-induced systemic immunosuppression and suggests that this pathway is likely to be an important sensor of UVA-induced damage to the skin. 24 hours after UVA, UVB and ssUV irradiation, a 1 cm2 uniform section of skin was excised from the dorsal surface of irradiated and control mice. Total RNA was then extracted from the whole skin using TRIzol reagent (Gibco Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturerâ??s instructions, purified, DNase treated and reverse transcribed into cDNA. For the microarray study a direct incorporation of Cyanine 3-dCTP and Cyanine 5-dCTP fluorescent dyes (Perkin Elmer Life Sciences, Inc. Boston, MA, USA) was used for cDNA synthesis. For each UV dose, a reference design was used to compare an unirradiated control against an irradiated sample. Microarray experiments used compugen 22k mouse oligonucleotide microarray slides (The Clive and Vera Ramaciotti Centre for Gene Function Analysis, Sydney Australia (http://www.ramaciotti.unsw.edu.au). Lower and higher UVA doses were used. C57BL/6 mice were irradiated with lower UVA, higher UVA, UVB, or ssUV; Balb/C mice were irradiated with lower or higher UVA. Experiments were replicated 6 times for each UV dose. A fluorescent dye swap was done for each alternate hybridisation to reduce systematic dye bias of incorporated fluorescent dyes.

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Small Perturbation Approach Reveals Transcriptomic Steady State

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