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2.  Effective Alu Repeat Based RT-Qpcr Normalization in Cancer Cell Perturbation Experiments 
PLoS ONE  2013;8(8):e71776.
Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.
The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5).
We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.
PMCID: PMC3743747  PMID: 23977142
3.  Dynamic Activity of miR-125b and miR-93 during Murine Neural Stem Cell Differentiation In Vitro and in the Subventricular Zone Neurogenic Niche 
PLoS ONE  2013;8(6):e67411.
Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function.
PMCID: PMC3694868  PMID: 23826292
4.  CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression 
PLoS ONE  2013;8(4):e60275.
Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.
PMCID: PMC3613353  PMID: 23560086
5.  Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes 
PLoS ONE  2013;8(1):e52321.
Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.
PMCID: PMC3537730  PMID: 23308108
6.  LNCipedia: a database for annotated human lncRNA transcript sequences and structures 
Nucleic Acids Research  2012;41(D1):D246-D251.
Here, we present LNCipedia (, a novel database for human long non-coding RNA (lncRNA) transcripts and genes. LncRNAs constitute a large and diverse class of non-coding RNA genes. Although several lncRNAs have been functionally annotated, the majority remains to be characterized. Different high-throughput methods to identify new lncRNAs (including RNA sequencing and annotation of chromatin-state maps) have been applied in various studies resulting in multiple unrelated lncRNA data sets. LNCipedia offers 21 488 annotated human lncRNA transcripts obtained from different sources. In addition to basic transcript information and gene structure, several statistics are determined for each entry in the database, such as secondary structure information, protein coding potential and microRNA binding sites. Our analyses suggest that, much like microRNAs, many lncRNAs have a significant secondary structure, in-line with their presumed association with proteins or protein complexes. Available literature on specific lncRNAs is linked, and users or authors can submit articles through a web interface. Protein coding potential is assessed by two different prediction algorithms: Coding Potential Calculator and HMMER. In addition, a novel strategy has been integrated for detecting potentially coding lncRNAs by automatically re-analysing the large body of publicly available mass spectrometry data in the PRIDE database. LNCipedia is publicly available and allows users to query and download lncRNA sequences and structures based on different search criteria. The database may serve as a resource to initiate small- and large-scale lncRNA studies. As an example, the LNCipedia content was used to develop a custom microarray for expression profiling of all available lncRNAs.
PMCID: PMC3531107  PMID: 23042674
7.  Accurate RT-qPCR gene expression analysis on cell culture lysates 
Scientific Reports  2012;2:222.
Gene expression quantification on cultured cells using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) typically involves an RNA purification step that limits sample processing throughput and precludes parallel analysis of large numbers of samples. An approach in which cDNA synthesis is carried out on crude cell lysates instead of on purified RNA samples can offer a fast and straightforward alternative. Here, we evaluate such an approach, benchmarking Ambion's Cells-to-CT kit with the classic workflow of RNA purification and cDNA synthesis, and demonstrate its good accuracy and superior sensitivity.
PMCID: PMC3257325  PMID: 22355736
8.  The miR-17-92 microRNA cluster regulates multiple components of the TGFβ-pathway in neuroblastoma 
Molecular cell  2010;40(5):762-773.
The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGFβ-signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGFβ-signaling cascade as well as through direct inhibition of TGFβ-responsive genes.
PMCID: PMC3032380  PMID: 21145484
9.  The microRNA body map: dissecting microRNA function through integrative genomics 
Nucleic Acids Research  2011;39(20):e136.
While a growing body of evidence implicates regulatory miRNA modules in various aspects of human disease and development, insights into specific miRNA function remain limited. Here, we present an innovative approach to elucidate tissue-specific miRNA functions that goes beyond miRNA target prediction and expression correlation. This approach is based on a multi-level integration of corresponding miRNA and mRNA gene expression levels, miRNA target prediction, transcription factor target prediction and mechanistic models of gene network regulation. Predicted miRNA functions were either validated experimentally or compared to published data. The predicted miRNA functions are accessible in the miRNA bodymap, an interactive online compendium and mining tool of high-dimensional newly generated and published miRNA expression profiles. The miRNA bodymap enables prioritization of candidate miRNAs based on their expression pattern or functional annotation across tissue or disease subgroup. The miRNA bodymap project provides users with a single one-stop data-mining solution and has great potential to become a community resource.
PMCID: PMC3203610  PMID: 21835775
10.  Chromosomal and miRNA Expression Patterns Reveal Biologically Distinct Subgroups of 11q− Neuroblastoma 
The purpose of this study was to further define the biology of the 11q− neuroblastoma tumor subgroup by the integration of aCGH with miRNA expression profiling data to determine if improved patient stratification is possible.
Experimental Design
A set of primary neuroblastoma (n=160) which was broadly representative of all genetic subtypes was analyzed by aCGH and for the expression of 430 miRNAs. A 15 miRNA expression signature previously demonstrated to be predictive of clinical outcome was used to analyze an independent cohort of 11q− tumors (n=37).
Loss of 4p and gain of 7q occurred at a significantly higher frequency in the 11q−tumors, further defining the genetic characteristics of this subtype. The 11q− tumors could be split into two subgroups using a miRNA expression survival signature which differed significantly in both clinical outcome and the overall frequency of large scale genomic imbalances, with the poor survival subgroup having significantly more imbalances. MiRNAs from the expression signature which were up-regulated in unfavorable tumors were predicted to target down-regulated genes from a published mRNA expression classifier of clinical outcome at a higher than expected frequency, indicating the miRNAs might contribute to the regulation of genes within the signature.
We demonstrate that two distinct biological subtypes of neuroblastoma with loss of 11q occur which differ in their miRNA expression profiles, frequency of segmental imbalances and clinical outcome. A miRNA expression signature, combined with an analysis of segmental imbalances, provides greater prediction of EFS and OS outcomes than 11q status by itself, improving patient stratification.
PMCID: PMC2880207  PMID: 20406844
aCGH; MYCN; neuroblastoma; miRNA
11.  Monoallelic but not bialleleic loss of Dicer1 promotes tumorigenesis in vivo 
Cell death and differentiation  2009;17(4):633-641.
Human tumors are characterized by widespread reduction in microRNA (miRNA) expression [1], although it is unclear how such changes come about and whether they have an etiological role in the disease. Importantly, miRNA-knockdown has been shown to enhance the tumorigenic potential of human lung adenocarcinoma cells [2]. A defect in miRNA-processing is one possible mechanism for the global down-regulation. To explore this possibility in more detail in vivo we have manipulated Dicer1 gene dosage in a mouse model of retinoblastoma. We show that while monoallelic loss of Dicer1 does not affect normal retinal development it dramatically accelerates tumor formation on a retinoblastoma-sensitized background. Importantly, these tumors retain one wild-type Dicer1 allele and exhibit only partial decrease in miRNA-processing. Accordingly, in silico analysis of human cancer genome data reveals frequent hemizygous, but not homozygous, deletions of DICER1. Strikingly, complete loss of Dicer1 function in mice did not accelerate retinoblastoma formation. miRNA profiling of these tumors identified members of the let-7 and miR-34 families as candidate tumor suppressors in retinoblastoma. We conclude that Dicer1 functions as a haploinsufficient tumor suppressor. This finding has implications for cancer aetiology and cancer therapy.
PMCID: PMC2892162  PMID: 20019750
Dicer; microRNA; retinoblastoma; tumor suppressor; haploinsufficiency
12.  miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis 
Nature cell biology  2010;12(3):247-256.
MicroRNAs (miRNAs) are increasingly implicated in regulating the malignant progression of cancer. Here we show that miR-9, the level of which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding mRNA, leading to increased cell motility and invasiveness. miR-9-mediated E-cadherin downregulation results in the activation of β-catenin signaling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumor angiogenesis. Overexpression of miR-9 in otherwise-non-metastatic breast tumor cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 using a ‘miRNA sponge’ in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumor grade, and metastatic status. These findings uncover a regulatory and signaling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin.
PMCID: PMC2845545  PMID: 20173740
13.  The emerging molecular pathogenesis of neuroblastoma: implications for improved risk assessment and targeted therapy 
Genome Medicine  2009;1(7):74.
Neuroblastoma is one of the most common solid tumors of childhood, arising from immature sympathetic nervous system cells. The clinical course of patients with neuroblastoma is highly variable, ranging from spontaneous regression to widespread metastatic disease. Although the outcome for children with cancer has improved considerably during the past decades, the prognosis of children with aggressive neuroblastoma remains dismal. The clinical heterogeneity of neuroblastoma mirrors the biological and genetic heterogeneity of these tumors. Ploidy and MYCN amplification have been used as genetic markers for risk stratification and therapeutic decision making, and, more recently, gene expression profiling and genome-wide DNA copy number analysis have come into the picture as sensitive and specific tools for assessing prognosis. The applica tion of new genetic tools also led to the discovery of an important familial neuroblastoma cancer gene, ALK, which is mutated in approximately 8% of sporadic tumors, and genome-wide association studies have unveiled loci with risk alleles for neuroblastoma development. For some of the genomic regions that are deleted in some neuroblastomas, on 1p, 3p and 11q, candidate tumor suppressor genes have been identified. In addition, evidence has emerged for the contribution of epigenetic disturbances in neuroblastoma oncogenesis. As in other cancer entities, altered microRNA expression is also being recognized as an important player in neuroblastoma. The recent successes in unraveling the genetic basis of neuroblastoma are now opening opportunities for development of targeted therapies.
PMCID: PMC2717400  PMID: 19638189
14.  Deep sequencing reveals differential expression of microRNAs in favorable versus unfavorable neuroblastoma 
Nucleic Acids Research  2010;38(17):5919-5928.
Small non-coding RNAs, in particular microRNAs(miRNAs), regulate fine-tuning of gene expression and can act as oncogenes or tumor suppressor genes. Differential miRNA expression has been reported to be of functional relevance for tumor biology. Using next-generation sequencing, the unbiased and absolute quantification of the small RNA transcriptome is now feasible. Neuroblastoma(NB) is an embryonal tumor with highly variable clinical course. We analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads. MiRNA expression profiles obtained by deep sequencing correlated well with real-time PCR data. Cluster analysis differentiated between favorable and unfavorable NBs, and the miRNA transcriptomes of these two groups were significantly different. Oncogenic miRNAs of the miR17-92 cluster and the miR-181 family were overexpressed in unfavorable NBs. In contrast, the putative tumor suppressive microRNAs, miR-542-5p and miR-628, were expressed in favorable NBs and virtually absent in unfavorable NBs. In-depth sequence analysis revealed extensive post-transcriptional miRNA editing. Of 13 identified novel miRNAs, three were further analyzed, and expression could be confirmed in a cohort of 70 NBs.
PMCID: PMC2943620  PMID: 20466808
15.  MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer 
BMC Cancer  2010;10:173.
Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies.
We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed.
In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue.
Our study demonstrates that the top six most stably expressed miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) described herein should be validated as suitable reference genes in both high-throughput and lower throughput RT-qPCR colorectal miRNA studies.
PMCID: PMC2873395  PMID: 20429937
16.  Widespread Dysregulation of MiRNAs by MYCN Amplification and Chromosomal Imbalances in Neuroblastoma: Association of miRNA Expression with Survival 
PLoS ONE  2009;4(11):e7850.
MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.
PMCID: PMC2773120  PMID: 19924232
17.  External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data 
Nucleic Acids Research  2009;37(21):e138.
The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.
PMCID: PMC2790878  PMID: 19734345
18.  A novel and universal method for microRNA RT-qPCR data normalization 
Genome Biology  2009;10(6):R64.
The mean expression value: a new method for accurate and reliable normalization of microRNA expression data from RT-qPCR experiments.
Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.
PMCID: PMC2718498  PMID: 19531210
19.  High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA 
Nucleic Acids Research  2008;36(21):e143.
MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.
PMCID: PMC2588502  PMID: 18940866

Results 1-19 (19)