Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

Radke, Lars; Giese, Christoph; Lubitz, Annika; Hinderlich, Stephan; Sandig, Grit; Hummel, Michael; Frohme, Marcus

doi:10.1007/s00604-014-1221-x

Cited by 10 publications

(9 citation statements)

References 25 publications

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“…The up-regulated genes participate in the biological functions such as regulation of cell proliferation, regulation of cell activity, cell migration and anti-apoptosis. In a recent report, the reference gene stability in peripheral blood mononuclear cells was determined by qPCR and NanoString [32]. In the present investigation, we have used the microarray technique.…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, 72 genes were identified that significantly differentially up-regulated by at least Cthreefold after the interaction with hybrid nanomatrix. The up-regulated genes were grouped into categories according to the biological functions such as regulation of cell proliferation (32), regulation of cell activity (12), cell migration (11) and anti-apoptosis (17) ( Table 2). Our observation on the basis of microarray was that the interaction of satellite cells with hybrid nanotextured matrix activates the expression of many genes mainly proliferation regulating genes (32 genes) and cell adhesion genes.…”

Section: Resultsmentioning

confidence: 99%

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Impact on gene expression in response to silver-decorated titania nanomatrix using an in vitro satellite cell culture model

et al. 2015

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Herein we report the synthesis of novel silver-ornamented titanium dioxide crossbreed nanofibers via electrospinning with improved cellular response for possible muscle tissue engineering applications. Titanium isopropoxide and silver nitrate served as precursors to get high aspect ratio silver-decorated titania nanofibers. For a model application in muscle tissue engineering, muscle satellite cells of local Korean Hanwoo were cultured on synthesized hybrid nanofibers. The standard microarray technique has been employed to screen the potential effects of synthesized nanofibers on genome after the culture of cells on hybrid nanofibers. EDX, EPMA, FT-IR, XRD and TEM analysis confirmed purity, distribution, interaction and crystalline nature of this material while the diameter of these nanofibers estimated from FE-SEM and TEM were between 200 and 300 nm. Cell counting with Kit-8 assay at regular time intervals and phase contrast microscopy data revealed that satellite cells proliferated well on nanofibers and cellular attachments were visible by SEM. Microarray analyses of gene expression response indicated clear changes in gene expression in a range of proliferation and apoptosis. Our results open-up new ways to develop and analyze gene expression biomarkers for satellite cell proliferation and finally muscle tissue engineering.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Impact on gene expression in response to silver-decorated titania nanomatrix using an in vitro satellite cell culture model

et al. 2015

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“…This eliminates the need to enzymatically amplify the template and also minimises pipetting procedures to reduce errors and contaminations. The platform reliably captures gene expression profiles effectively with relatively small amounts of template RNA (100 ng) and also serve as an alternative to RT-qPCR gene expression assays ( Radke et al, 2014 ; Hu et al, 2016 ). Previous cynomolgus macaque studies utilised RT-PCR and RT-qPCR to validate their high-throughput sequencing results, and as such, the present study is the first to validate cynomolgus macaque RNA-Seq data using the NanoString nCounter XT gene expression assay.…”

Section: Resultsmentioning

confidence: 99%

RNA sequencing (RNA-Seq) of lymph node, spleen, and thymus transcriptome from wild Peninsular Malaysian cynomolgus macaque (Macaca fascicularis)

Uli

Yong

Yeap

et al. 2017

PeerJ

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The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53–63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development.

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“…RNA Quality Numbers (RQN) were determined on a Fragment Analyzer ™ (Advanced Analytical Technologies, Heidelberg, Germany) with a DNF-472 High Sensitivity RNA Analysis Kit, using extended runtimes of 60 min per sample to be able to detect any remaining genomic DNA contaminations. The nCounter technology [ 28 ] is a multiplexed method that quantifies mRNA on single molecule level by using fluorescent barcoding probes and is described in detail in [ 29 ]. NanoString experiments were performed according to the manufacturer protocol.…”

Section: Methodsmentioning

confidence: 99%

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans

et al. 2017

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Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

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Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

Cited by 10 publications

References 25 publications

Impact on gene expression in response to silver-decorated titania nanomatrix using an in vitro satellite cell culture model

Impact on gene expression in response to silver-decorated titania nanomatrix using an in vitro satellite cell culture model

RNA sequencing (RNA-Seq) of lymph node, spleen, and thymus transcriptome from wild Peninsular Malaysian cynomolgus macaque (Macaca fascicularis)

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans

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