Gene Expression Analysis in Platelets from a Single Donor: Evaluation of a PCR-Based Amplification Technique

Rox, Jutta M.; Bugert, Peter; Müller, Jens; Schorr, Alexander; Hanfland, P.; Madlener, Katharina; Klüter, Harald; Pötzsch, Bernd

doi:10.1373/clinchem.2004.035386

Cited by 48 publications

(42 citation statements)

References 28 publications

(27 reference statements)

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“…The development of PCR and its application to platelet biology facilitated the characterization of platelet transcripts and was instrumental in defining many monogenic platelet disorders and the human platelet antigens (2,13,14). Recently, transcription profiling methods, such as serial analysis of gene expression (SAGE) and microarray technology, have been applied to more fully characterize transcripts in platelets (15)(16)(17)(18).…”

Section: Genomics Without a Genome: The Platelet Transcriptomementioning

confidence: 99%

“…Given the availability of whole-genome transcriptome platforms, robust RNA amplification techniques (15), and novel proteomics technologies, platelet biologists are now in a position to begin characterizing the full complement of transcripts and functionally relevant protein fractions in an individual's platelets. This allows a shift in focus away from determining the components of the platelet system to asking fundamental questions about the mechanisms that determine interindividual variation in platelet function.…”

Section: Platelet Proteomics and Genomics In Complex Diseasementioning

confidence: 99%

See 1 more Smart Citation

Platelet genomics and proteomics in human health and disease

Macaulay

Carr²,

Gusnanto³

et al. 2005

Journal of Clinical Investigation

150

100

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Proteomic and genomic technologies provide powerful tools for characterizing the multitude of events that occur in the anucleate platelet. These technologies are beginning to define the complete platelet transcriptome and proteome as well as the protein-protein interactions critical for platelet function. The integration of these results provides the opportunity to identify those proteins involved in discrete facets of platelet function. Here we summarize the findings of platelet proteome and transcriptome studies and their application to diseases of platelet function.

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Section: Genomics Without a Genome: The Platelet Transcriptomementioning

confidence: 99%

Section: Platelet Proteomics and Genomics In Complex Diseasementioning

confidence: 99%

Platelet genomics and proteomics in human health and disease

Macaulay

Carr²,

Gusnanto³

et al. 2005

Journal of Clinical Investigation

150

100

View full text Add to dashboard Cite

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“…The performance of SMART has been evaluated with cDNA-spotted (12,18 ) and oligonucleotide-spotted microarrays (19 ). Starting from low nanogram amounts of RNA, we previously used real-time PCR to determine the amplification factors of several target sequences and found a remarkably higher power of amplification for SMART PCR than for IVT (10 ).…”

mentioning

confidence: 99%

Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments

et al. 2006

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Background: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. Methods: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART TM for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomerspotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. Results: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. Conclusions: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is

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“…Moreover, RUNX1 interacts with GATA-1 and enforced RUNX1 expression in K562 cells enhances induction of megakaryocyticspecific integrin ␣IIb, suggesting an important RUNX1 role in megakaryocytic lineage commitment. 27 Because of altered megakaryopoiesis associated with RUNX1 mutations, 5 the contribution of 12-LO to megakaryocyte biology needs to be explored.Several studies have established the feasibility of platelet expression profiling [43][44][45] despite the limited amount of mRNA present in these anucleate cells. Our present studies provide further proof of concept that this technology can indeed elucidate specific and novel molecular aberrations in patients with inherited platelet dysfunction, in the majority of whom we currently have no understanding of the molecular mechanisms.…”

mentioning

confidence: 99%

RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency

Kaur

Jalagadugula

Mao

et al. 2010

Blood

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Haploinsufficiency of RUNX1 (also known as CBFA2/AML1) is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. We have reported on a patient with thrombocytopenia and impaired agonistinduced aggregation, secretion, and protein phosphorylation associated with a RUNX1 mutation. Expression profiling of platelets revealed approximately 5-fold decreased expression of 12-lipoxygenase (12-LO, gene ALOX12), which catalyzes 12-hydroxyeicosatetraenoic acid production from arachidonic acid. We hypothesized that ALOX12 is a direct transcriptional target gene of RUNX1. In present studies, agonist-induced platelet 12-HETE production was decreased in the patient. Four RUNX1 consensus sites were identified in the 2-kb promoter region of ALOX12 (at ؊1498, ؊1491, ؊708, ؊526 from ATG). In luciferase reporter studies in human erythroleukemia cells, mutation of each site decreased activity; overexpression of RUNX1 up-regulated promoter activity, which was abolished by mutation of RUNX1 sites. Gel shift studies, including with recombinant protein, revealed RUNX1 binding to each site. Chromatin immunoprecipitation revealed in vivo RUNX1 binding in the region of interest. siRNA knockdown of RUNX1 decreased RUNX1 and 12-LO proteins. ALOX12 is a direct transcriptional target of RUNX1. Our studies provide further proof of principle that platelet expression profiling can elucidate novel alterations in platelets with inherited dysfunction. (Blood. 2010;115(15):3128-3135) Introduction RUNX1, also known as CBFA2 (core binding factor A2), is a member of a family of transcription factors that regulate the expression of several hematopoietic-specific genes through a highly conserved DNA-binding region called the RUNT homology domain (RHD). 1 The RHD dimerizes with CBF␤ to form a stable complex. The complex acts as an anchor to recruit other cofactors that bind in cis to adjacent sites or interact directly with RUNX1. RUNX1 plays a critical role in normal fetal hematopoiesis. 2,3 Homozygous deletion of RUNX1 results in embryonic lethality related to absence of definitive hematopoiesis. [2][3][4] In humans, haploinsufficiency of RUNX1 is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. 5 Most of the point mutations identified in RUNX1 occur in the RHD leading to loss of DNA binding. 6,7 We have previously reported 8,9 studies in a patient with mild thrombocytopenia, impaired agonist-induced platelet aggregation, secretion and protein phosphorylation (myosin light chain and pleckstrin), and decreased platelet protein kinase C-(PKC-), associated with a mutation (haplodeficiency) in the conserved region of RUNX1. Expression profiling of patient platelets revealed an approximately 5-fold decreased mRNA expression of platelet-type 12-lipoxygenase (12-LO, gene ALOX12). 10 Lipoxygenases are a family of non-heme iron-containing enzymes that catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids, such as arachidonic acid (AA). Th...

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Gene Expression Analysis in Platelets from a Single Donor: Evaluation of a PCR-Based Amplification Technique

Cited by 48 publications

References 28 publications

Platelet genomics and proteomics in human health and disease

Platelet genomics and proteomics in human health and disease

Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments

RUNX1/core binding factor A2 regulates platelet 12-lipoxygenase gene (ALOX12): studies in human RUNX1 haplodeficiency

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