Project description:The study aimed to comprehensively characterize human myoblastic cell line RCMH using using electron microscopic and proteomic approaches. Myoblastic cell lines can be useful to investigate the complex biochemical changes occuring under different conditions that reflect the physiological and pathophysiological mechanisms of muscle. So far, there are no suitable in vitro models of human muscle origin to study a variety of muscle related processes including responses to mechanical stress, EC-coupling and (ER-associated) myopathic disorders. Therefore, we characterized the human immortal myoblastic cell line RCMH and the results suggest RCMH as a suitable in vitro model for investigating human muscle related processes and disorders.
Project description:Global transcriptome analyses provide an excellent basis for the identification and definition of biomarkers with high relevance in infection processes, therapeutic intervention and protective immunity. The measurement applies three different state of the art transcriptomic technologies for global expression profiling to vaccine development. Different microarray platforms in conjunct to next generation sequencing (NGS) will build the basis for comparative approaches, such as up-down classification and correlation coefficients. This measurement is based on Agilent microarrays and a clinical trial phase Ia study with M. bovis BCG vaccination, using two different tuberculin skin test (PPD negative and PPD positive) groups. • Surrogate measurement using PBMCs • 4 time points: d0 (naïve, pre-immunization) and d29, d57, d180 post m. bovis BCG immunization • Responses of PPD negative and PPD positive study groups • Group size of approximately 9 individuals European network of vaccine research and development (TRANSVAC)
Project description:Global transcriptome analyses provide an excellent basis for the identification and definition of biomarkers with high relevance in infection processes, therapeutic intervention and protective immunity. The measurement applies three different state of the art transcriptomic technologies for global expression profiling to vaccine development. Different microarray platforms in conjunct to next generation sequencing (NGS) will build the basis for comparative approaches, such as up-down classification and correlation coefficients. This measurement is based on Agilent microarrays and a clinical trial phase Ia study with M. bovis BCG vaccination, using two different tuberculin skin test (PPD negative and PPD positive) groups. � Surrogate measurement using PBMCs � 4 time points: d0 (naïve, pre-immunization) and d29, d57, d180 post m. bovis BCG immunization � Responses of PPD negative and PPD positive study groups � Group size of approximately 9 individuals European network of vaccine research and development (TRANSVAC) Microarray experiments were performed as single-color hybridizations using Agilent Technologies whole human genome 4x44K microarrays
Project description:Global transcriptome analyses provide an excellent basis for the identification and definition of biomarkers with high relevance in infection processes, therapeutic intervention and protective immunity. The measurement applies three different state of the art transcriptomic technologies for global expression profiling to vaccine development. Different microarray platforms in conjunct to next generation sequencing (NGS) will build the basis for comparative approaches, such as up-down classification and correlation coefficients. This measurement is based on Agilent microarrays and a clinical trial phase Ib study with M. bovis BCG vaccination. • Surrogate measurement using whole human blood • 4 time points: d0 (naïve, pre-immunization) and d14, d28,d56, d168 post m. bovis BCG immunization • Responses of PPD positive study groups • Group size of approximately 6 individuals European network of vaccine research and development (TRANSVAC)
Project description:Global transcriptome analyses provide an excellent basis for the identification and definition of biomarkers with high relevance in infection processes, therapeutic intervention and protective immunity. The measurement applies three different state of the art transcriptomic technologies for global expression profiling to vaccine development. Different microarray platforms in conjunct to next generation sequencing (NGS) will build the basis for comparative approaches, such as up-down classification and correlation coefficients. This measurement is based on Agilent microarrays and a clinical trial phase Ib study with M. bovis BCG vaccination. M-bM-^@M-" Surrogate measurement using whole human blood M-bM-^@M-" 4 time points: d0 (naM-CM-/ve, pre-immunization) and d14, d28,d56, d168 post m. bovis BCG immunization M-bM-^@M-" Responses of PPD positive study groups M-bM-^@M-" Group size of approximately 6 individuals European network of vaccine research and development (TRANSVAC) Microarray experiments were performed as single-color hybridizations using Agilent Technologies whole human genome 4x44K microarrays
Project description:Deeper understanding of T cell biology is crucial for the development of new therapeutics. Human naïve T cells have low RNA content and their numbers can be limiting; therefore we set out to determine the parameters for robust ultra-low input RNA sequencing. We performed transcriptome profiling at different cell inputs and compared three protocols: Switching Mechanism at 5’ End of RNA Template technology (SMART) with two different library preparation methods (Nextera (SMART_Nxt) and Clontech (SMART_CC)), and AmpliSeq technology. As the cell input decreased the number of detected coding genes decreased with SMART, while stayed constant with AmpliSeq. However, SMART enables detection of non-coding genes, which is not feasible for AmpliSeq. The detection is dependent on gene abundance, but not transcript length. The consistency between technical replicates and cell inputs was comparable across methods above 1K but highly variable at 100 cell input. Sensitivity of detection for differentially expressed genes decreased dramatically with decreased cell inputs in all protocols, support that additional approaches, such as pathway enrichment, are important for data interpretation at ultra-low input. Finally, T cell activation signature was detected at 1K cell input and above in all protocols, with AmpliSeq showing better detection at 100 cells.
Project description:PURPOSE: To identify genes whose expressions in primary human trabecular meshwork (TM) cell cultures are affected by the transcription factor pituitary homeobox 2 (PITX2) and to identify genes that may have roles in glaucoma. Known glaucoma causing genes account for disease in a small fraction of patients, and we aimed at identification of other genes that may have subtle and accumulative effects not easily identifiable by a genetic approach. METHODS: Expression profiles derived using microarrays were compared between TM control cells and cells treated with PITX2 siRNAs using three protocols so as to minimize false positive and negative results. The first protocol was based on the commonly used B statistic. The second and third protocols were based on fold change in expression. The second protocol used a threshold of at least 2 fold change in expression, whereas the third protocol used ranking in fold change without setting a threshold. The likelihood of a selected gene being a true positive was considered to correlate with the number of protocols by which it was selected. By considering all genes that were selected by at least one protocol, the likelihood of false negatives was expected to decrease. Effects on a subset of selected genes were verified by real time PCR, western blots, and immunocytochemistry. Effects on ALDH1A1, were further pursued because its protein product, aldehyde dehydrogenase 1 family, member A1, has roles in oxidative stress and because oxidative stress is known to be relevant to the etiology of glaucoma. RESULTS: The expression level of 41 genes was assessed by to be possibly affected by PITX2 knockdown. Twenty one genes were down-regulated and twenty were upregulated. The expression of five genes was assessed to be altered by all three analysis protocols. The five genes were DIRAS3 (DIRAS family, GTP-binding RAS-like 3), CXCL6 (chemokine (C-X-C motif) ligand 6), SAMD5 (sterile alpha motif domain containing 5), CBFB (core-binding factor, beta subunit), and MEIS2 (meis homeobox 2). Real time PCR experiments verified results on a subset of genes tested. Notably, the results were also confirmed in two independent TMs. Effects on CXCL6 and ALDH1A1 were also confirmed by western blots, and effects on ALDH1A1 were further shown by immunocytochemistry. Data consistent with PITX2 involvement in ALDH1A1 mediated response to oxidative stress were presented. CONCLUSIONS: Bioinformatics tools revealed that the genes identified affect functions and pathways relevant to glaucoma. Involvement of PITX2 in expression of some of the genes and in some of the pathways is being reported here for the first time. As many of the genes identified have not been studied vis-à-vis glaucoma, we feel they introduce new candidates for understanding this devastating disease.