Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA

Mastrokolias, Anastasios; Dunnen, Johan T. den; Ommen, Gert‐Jan B. van; Hoen, Peter A C ‘t; Roon‐Mom, Willeke M. C. van

doi:10.1186/1471-2164-13-28

Cited by 62 publications

(81 citation statements)

References 30 publications

(34 reference statements)

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“…Their reproducible depletion across all samples suggests that the globin depletion procedure itself is responsible, though why these particular transcripts were selectively depleted is not known. Unexplained, reproducible depletion of non-globin transcripts following globin depletion has been previously reported [7]. Assessing the effects of globin depletion on well annotated transcripts, mapping to unique gene symbols only, yielded more predictable results: over a thousand genes were consistently up-regulated as a result of globin depletion (FDR≤0.01), and only a handful of genes were consistently down-regulated across various experimental designs.…”

Section: Discussionmentioning

confidence: 67%

Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

Shin¹,

Shannon

Fishbane

et al. 2014

PLoS ONE

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BackgroundThe molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples.ResultsWe compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding.ConclusionsGlobin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.

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Section: Discussionmentioning

confidence: 67%

Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

Shin¹,

Shannon

Fishbane

et al. 2014

PLoS ONE

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“…mRNA was profiled using Deep-SAGE sequencing using Illumina technology (Mastrokolias et al, 2012). CATG was added to the 5′ end of the 17 base pair sequences obtained.…”

Section: Gene Expressionmentioning

confidence: 99%

“…Expression of genes of interest was validated using independent biological samples by RT-PCR after generation of cDNA (cDNA synthesis kit, Roche, Basel, Switzerland) using the listed primers (Table S3), as previously described (Mastrokolias et al, 2012). Expression levels were calculated relative to expression of the housekeeping genes GAPDH and HPRT1.…”

Section: Gene Expressionmentioning

confidence: 99%

Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro

et al. 2015

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Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets.

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“…25 Data were mapped against the UCSChg19 reference genome using Bowtie, with permission of one mismatch and only retaining unique mappings.…”

Section: Gene Expressionmentioning

confidence: 99%

Gene-expression and in vitro function of mesenchymal stromal cells are affected in juvenile myelomonocytic leukemia

et al. 2015

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An aberrant interaction between hematopoietic stem cells and mesenchymal stromal cells has been linked to disease and shown to contribute to the pathophysiology of hematologic malignancies in murine models. Juvenile myelomonocytic leukemia is an aggressive malignant disease affecting young infants. Here we investigated the impact of juvenile myelomonocytic leukemia on mesenchymal stromal cells. Mesenchymal stromal cells were expanded from bone marrow samples of patients at diagnosis (n=9) and after hematopoietic stem cell transplantation (n=7; from 5 patients) and from healthy children (n=10). Cells were characterized by phenotyping, differentiation, gene expression analysis (of controls and samples obtained at diagnosis) and in vitro functional studies assessing immunomodulation and hematopoietic support. Mesenchymal stromal cells from patients did not differ from controls in differentiation capacity nor did they differ in their capacity to support in vitro hematopoiesis. Deep-SAGE sequencing revealed differential mRNA expression in patient-derived samples, including genes encoding proteins involved in immunomodulation and cell-cell interaction. Selected gene expression normalized during remission after successful hematopoietic stem cell transplantation. Whereas natural killer cell activation and peripheral blood mononuclear cell proliferation were not differentially affected, the suppressive effect on monocyte to dendritic cell differentiation was increased by mesenchymal stromal cells obtained at diagnosis, but not at time of remission. This study shows that active juvenile myelomonocytic leukemia affects the immune responserelated gene expression and function of mesenchymal stromal cells. In contrast, the differential gene expression of hematopoiesis-related genes could not be supported by functional data. Decreased immune surveillance might contribute to the therapy resistance and progression in juvenile myelomonocytic leukemia.

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Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA

Cited by 62 publications

References 30 publications

Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro

Gene-expression and in vitro function of mesenchymal stromal cells are affected in juvenile myelomonocytic leukemia

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