Jo Vandesompele

Bio: Jo Vandesompele is an academic researcher from Ghent University. The author has contributed to research in topics: Neuroblastoma & microRNA. The author has an hindex of 88, co-authored 383 publications receiving 59368 citations. Previous affiliations of Jo Vandesompele include Washington University in St. Louis & Ghent University Hospital.

Quantification and normalization of gene expression using SYBR Green I real-time RT-PCR

Abstract: s: Session III 9 2 van der Pluijm, Gabri [49] Monitoring metastatic behavior of human breast cancer cells in mice with speciesspecific PCR and bioluminescent reporter imaging G. van der Pluijm1, A. Wetterwald 2, B. Sijmons1, E. Gautschi2, I. Que1, B. Stadler3, G. Thalmann2, M. Cecchini2 & C. Löwik1 1Leiden University Medical Center, Dept. of Endocrinology, Leiden, The Netherlands 2Gene Therapy Laboratory, Department of Clinical Research and Urology Clinic, University Hospital, Bern, Switzerland 3Institute of Immunology and Allergology, University Hospital, Bern, Switzerland Breast cancer metastasizes frequently to the skeleton and causes considerable morbidity. Injection of human MDA-MB-231 breast cancer cells into the left heart ventricle of immunodeficient mice produced multiple osteolytic bone lesions and soft tissue metastases. However, micrometastases are not readily detectable and more sensitive methods are required to detect minimal disease states. Here we describe the use of species-specific PCR (ssPCR) and bioluminescent reporter imaging (BRI) for monitoring the tumor metastatic in vivo. We used a CCD camera connected to the Argus-20 image processor (C-2400/VIM, Hamamatsu) to monitor bone metastasis by tumor cells transfected with the luciferase reporter gene. MDA-MB-231 cells were stably transfected with a pCMV-plasmid containing the firefly luciferase gene (MDA-231/luc+). After intracardiac inoclation with MDA-231/luc+ cells, the mice were monitored weekly for the development of bone and soft tissue metastases. For this, the mice were anaesthesized, injected with D-luciferin and photon emission was measured. Micrometastases developed predominantly in the hind limbs and the axial skeleton. Distinct photon emission was detected already after 24 days in bone (detection limit ± 10.000 cells). ssPCR revealed significantly increased expression of VEGF-A, -B and PTHrP by the cancer cells in bone metastases when compared to soft tissues metastases suggesting a causal role in the observed preferential skeletal metastasis. In conclusion, BRI and ssPCR can be used for quantitative detection and growth of micrometastases and may become extremely useful to study the pathogenesis of bone metastasis in living animals, to monitor expression of targeted gene vectors and to determine the efficacy of novel therapeutic agents. Vandesompele, Jo [50] Integration of suppression subtractive hybridization, laser capture microdissection and microarray analysis for identification of genes implicated in neuroblastoma pathogenesis Jo Vandesompele1, Nadine Van Roy1, Geneviève Laureys2, Katleen De Preter1, Geert Berx3, Kristin Strumane3, Anne De Paepe1, Frans Van Roy3 & Frank Speleman1 1Center for Medical Genetics, University Hospital, Ghent, Belgium 2Department of Pediatric Oncology, University Hospital, Ghent, Belgium 3Department of Molecular Biology, VIB, Belgium Neuroblastoma is the most frequent extracranial solid tumor in children, showing remarkable clinical and genetic heterogeneity. To make further progress in understanding the genetic basis of neuroblastoma we have applied suppression subtractive hybridization for the identification of differentially expressed transcripts. We analyzed two neuroblastoma cell lines belonging to different genetic subgroups, respectively with and without 1p and 11q deletion and MYCN amplification. Northern blot analysis and real-time quantitative polymerase chain reaction with reverse transcription showed differential expression in 75% of the selected genes. We identified both rare and highly abundant differential transcripts. Known and previously unreported genes from the 2p23 and 2p13–14 amplified regions in IMR32 were efficiently selected. From the present set of suppression subtractive hybridization clones, we will select genes relevant to normal neurogenesis through expression analysis of normal fetal neuroblasts isolated by laser capture microdissection (Arcturus, PixCell II). We will select genes implicated in particular subsets of neuroblastoma by microarray analysis (Affymetrix 417) of a large panel of neuroblastoma cell lines and primary tumors. Vandesompele, Jo [51] Quantification and normalization of gene expression using SYBR Green I real-time RT-PCR Jo Vandesompele, Johanna Iso-Oja, Anne De Paepe & Frank Speleman Center for Medical Genetics 1K5, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium We have developed a two-step, real-time, quantitative assay, using the polymerase chain reaction with reverse transcription (RT-PCR) and based on SYBR Green I monitoring of PCR product accumulation, for quantification and normalization of gene expression levels. Because housekeeping gene expression can vary considerably among cell types or experimental conditions, our procedure uses multiple internal control genes for more accurate normalization of expression data, compared with traditional use of only one housekeeping gene. Owing to extensive accumulation of primer-dimers when no template control is used during one-step RT-PCR, we introduced a two-step protocol to eliminate this problem. This study further illustrates the prerequisite of DNase treatment of RNA samples before complementary DNA synthesis. The treatment also results in a significantly facilitated primer design for RT-PCR, as the positions of the primers are no longer important to control for genomic contamination. Real-time quantitative RT-PCR of DNase-treated samples and normalization using multiple internal controls is the method of choice for sensitive, accurate and large-scale measurements of gene expression levels.

A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma

Abstract: Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Using real-time qPCR to evaluate RNAi-mediated gene silencing

Abstract: 39 Protocol Guide | 2010 Introduction Neuroblastoma is a childhood cancer that is derived from precursor cells of the adrenosympathetic system, originating in the adrenal medulla or sympathetic ganglia. To gain insight into the mechanism of action of the MYCN gene in neuroblastoma pathogenesis, we used RNA interference (RNAi) to suppress MYCN expression. The MYCN gene was transiently suppressed using 27-mer siLentMerTM* Dicer-substrate small-interfering RNA (siRNA) duplexes (Bio-Rad Laboratories, Inc.), which are generally considered more ef fective at gene silencing than their corresponding traditional 21-mer siRNAs (1). The CFX96TM real-time PCR detection system (Bio-Rad Laboratories, Inc.) was used to monitor silencing efficiency and assess functional effects of RNAi-mediated knockdown of MYCN.

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

Abstract: Background The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulfite-based cfDNA methylation profiling. Methods 15 tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulfite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. Results All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. Conclusion The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients

Abstract: In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of non-invasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in two independent cohorts of 131 and 54 patients. Evaluation of serum miRNA variance in a model testing for tumor stage, MYCN status, age at diagnosis and overall survival, revealed tumor stage as the most significant factor impacting miRNA abundance in neuroblastoma serum. Differential expression analysis between patients with metastatic and localized disease revealed 9 miRNAs strongly associated with metastatic stage 4 disease in both patient cohorts. Increasing levels of these miRNAs were also observed in serum from xenografted mice bearing human neuroblastoma tumors. Moreover, murine serum miRNA levels were strongly associated with tumor volume, suggesting this miRNA signature may be applied to monitor disease burden.

limma powers differential expression analyses for RNA-sequencing and microarray studies

Abstract: limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.

Cutadapt removes adapter sequences from high-throughput sequencing reads

Abstract: When small RNA is sequenced on current sequencing machines, the resulting reads are usually longer than the RNA and therefore contain parts of the 3' adapter. That adapter must be found and removed error-tolerantly from each read before read mapping. Previous solutions are either hard to use or do not offer required features, in particular support for color space data. As an easy to use alternative, we developed the command-line tool cutadapt, which supports 454, Illumina and SOLiD (color space) data, offers two adapter trimming algorithms, and has other useful features. Cutadapt, including its MIT-licensed source code, is available for download at http://code.google.com/p/cutadapt/

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

Abstract: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.