Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

Çınar, Mehmet Ulaş; Islam, Mohammad Ariful; Pröll, Maren; Kocamiş, Hakan; Tholen, Ernst; Tesfaye, Dawit; Looft, Christian; Schellander, K.; Uddin, Muhammad Jasim

doi:10.1186/1756-0500-6-56

Cited by 39 publications

(39 citation statements)

References 49 publications

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“…Clean reads quality metrics are shown in (Table 1). We used StringTie [12] to reconstruct transcripts, then we used Cuffmerge (Cufflinks tools [13]) to bring together the refactoring information for all our samples, and used Cuffcompare to compare reconstructed transcripts to reference annotation. Subsequently, we mapped clean reads to a reference using Bowtie 2 [14], followed by the gene expression level calculation using RSEM [15].…”

Section: Rnaseq Data Analysesmentioning

confidence: 99%

“…To validate the accuracy of the DGE result, 20 of biologically relevant genes from the same RNA used for DGEs sequencing were randomly selected. Primer premier 5.0 was used to design the targeted genes primers ( Supplementary Table 1) and the internal reference gene (GAPDH) was selected based on the published reference gene GAPDH (Genbank: 396823) [12].…”

Section: Genes Expression Analysis By Qrt-pcrmentioning

confidence: 99%

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Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets

Sun

Fahd

et al. 2019

Microbial Pathogenesis

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A B S T R A C TCaused by porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PED) is an acute infectious disease which causes damage to the intestine including intestinal villus atrophy and shedding, leading to serious economic losses to the pig industry worldwide. In order to obtain detailed information about the pathogenesis and host immune response in a PEDV-infected host for first In vivo study we used high-throughput sequencing to analyze the gene expression differences of the small intestinal mucosa after infection with PEDV. Transcripts obtained were over 65,525,000 clean reads after reassembly were 22,605 genes detected, of which 22,248 were known genes and 371 new genes were predicted. Moreover, 3168 genes expression was up-regulated and 3876 genes down-regulated. (Gene Ontology) GO annotation and functional enrichment analysis indicated that all of the DEGs (differentially expressed genes) were annotated into biological process, cellular component and molecular function. Most of these unigenes are annotated in cellular processes, the cell and binding. KEGG analysis of the DEGs showed that a total of 7044 DEGs unigenes were annotated into 323 pathways classified into 6 main categories. Most of these unigenes are annotated were related to immune system response to the infectious diseases pathways. In addition, 20 DEGs were verified by quantitative real-time PCR. As the first, in vivo, RNAseq analysis of piglets and PEDV infection, our study provides knowledge about the transcriptomics of intestinal mucosa in PEDV-infected piglets, from which a complex molecular pathways and pathogenesis-related biological processes are involved in PEDV interaction with piglet intestinal mucosa.

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Section: Rnaseq Data Analysesmentioning

confidence: 99%

Section: Genes Expression Analysis By Qrt-pcrmentioning

confidence: 99%

Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets

Sun

Fahd

et al. 2019

Microbial Pathogenesis

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“…This is reasonable, as from the host's point of a view an invading microorganism is simply undesirable, whatever its reaction to the Gram stain. Indeed, modulo a few detailed differences [30], and certainly for our present purposes, it seems that LTA is indeed broadly equivalent to LPS in terms of its ability to stimulate an innate immune response [31][32][33].…”

Section: Introductionmentioning

confidence: 77%

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker^™stains

Pretorius

Pagé

Hendricks

et al. 2017

Preprint

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In recent work, we discovered that the presence of highly substoichiometric amounts (10 -8 molar ratio) of lipopolysaccharide (LPS) from Gram-negative bacteria caused fibrinogen clotting to lead to the formation of an amyloid form of fibrin. We here show that the broadly equivalent lipoteichoic acids (LTAs) from two species of Gram-positive bacteria have similarly (if not more) potent effects. Using thioflavin T fluorescence to detect amyloid as before, the addition of low concentrations of free ferric ion is found to have similar effects. Luminescent conjugated oligothiophene dyes (LCOs), marketed under the trade name Amytracker TM , also stain classical amyloid structures. We here show that they too give very large fluorescence enhancements when clotting is initiated in the presence of the four amyloidogens (LPS, ferric ions and two LTA types). The staining patterns differ significantly as a function of both the amyloidogens and the dyes used to assess them, indicating clearly that the nature of the clots formed is different. This is also the case when clotting is measured viscometrically using thermoelastography. Overall, the data provide further evidence for an important role of bacterial cell wall products in the various coagulopathies that are observable in chronic, inflammatory diseases. The assays may have potential in both diagnostics and therapeutics.

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“…Using these statistical algorithms, a number of suitable reference miRNAs have been identified for quantifying miRNAs expression in humans (Doyle et al, 2002;Pfaffl et al, 2004;Guenin et al, 2009;Shen et al, 2011;Lamba et al, 2014) and in livestock species (Gu et al, 2011;Wessels et al, 2011;Timoneda et al, 2012;Li et al, 2014). Previous studies suggested that different mRNA reference genes are required for mRNA gene expression studies in porcine PBMCs at different stages or with different immunological stimulants (Ju et al, 2011;Cinar et al, 2013). While many reference genes suitable for mRNA expression studies have been identified in porcine PBMCs, no well-established reference miRNAs have been investigated.…”

Section: Discussionmentioning

confidence: 98%

Identification of reference microRNAs for quantitative expression analysis in porcine peripheral blood mononuclear cells treated with polyinosinic–polycytidylic acid

Wang

Sun

et al. 2015

Int J Immunogenetics

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Peripheral blood mononuclear cells (PBMCs) are clinically important cells. Detection of microRNAs (miRNAs) expression in PBMCs can be useful for miRNA biomarker discovery for various diseases. Quantitative real-time PCR (qRT-PCR) has become an important method used for measuring miRNAs expression. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. Here, we performed qRT-PCR to quantify the expression levels of nine miRNAs (Ssc-miR-16, Hsa-miR-25, Ssc-miR-34a, Hsa-miR-93, Bta-miR-92b, Ssc-miR-103, Ssc-miR-106a, Ssc-miR-128 and Ssc-miR-107) and one small nuclear RNA (U6) in PBMCs treated with polyinosinic-polycytidylic acid [poly (I:C)] that widely used for simulating viral infection. We used the four statistical algorithms (GeNorm 3.5, NormFinder, BestKeeper and comparative ∆ Ct method) to evaluate gene expression stability and observed that Ssc-miR-34a was the best single reference gene and the pair of Ssc-miR-107 and Ssc-miR-103 was the best combination of reference miRNAs for porcine PBMCs treated with poly (I:C). Our study shows the first evidence of careful selection of reference miRNAs in porcine PBMCs and maybe helpful for discovering miRNA biomarkers for double-stranded RNA-induced disease.

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Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

Cited by 39 publications

References 49 publications

Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets

Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker^™stains

Identification of reference microRNAs for quantitative expression analysis in porcine peripheral blood mononuclear cells treated with polyinosinic–polycytidylic acid

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Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

Cited by 39 publications

References 49 publications

Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets

Transcriptomic analysis of small intestinal mucosa from porcine epidemic diarrhea virus infected piglets

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™stains

Identification of reference microRNAs for quantitative expression analysis in porcine peripheral blood mononuclear cells treated with polyinosinic–polycytidylic acid

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Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker^™stains