Showing papers by "Jo Vandesompele published in 2009"

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

A novel and universal method for microRNA RT-qPCR data normalization

Abstract: 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.

Amphiregulin and epiregulin mRNA expression in primary tumors predicts outcome in metastatic colorectal cancer treated with cetuximab

Abstract: PURPOSE: To study the power of the epidermal growth factor receptor (EGFR) epiregulin (EREG) and amphiregulin (AREG) ligands' expression in primary tumors to predict the outcome in patients with chemorefractory metastatic colorectal cancer (cmCRC) treated with the combination of cetuximab and irinotecan. PATIENTS AND METHODS: Gene expression measurements and KRAS mutation analysis were performed on archival formalin-fixed paraffin-embedded primary tumors of 220 cmCRC patients. Response was measured using RECIST (Response Evaluation Criteria in Solid Tumors) criteria. The relation between ligand expression levels and outcome was evaluated using logistic regression for response and Cox regression for survival data. Receiver operating characteristics analysis was performed for response and survival data. CIs for the performance indices were obtained with a nonparametric bootstrap procedure. Findings were externally validated on a series of 67 samples treated in a similar setting. RESULTS: In KRAS wild type (WT) patients, there was a significant association between log-transformed ligand expression and response for EREG (odds ratio for objective response, 1.90; 95% CI, 1.27 to 2.83; P = .0005; concordance index [c-index], 0.681) and for AREG (odds ratio for objective response, 1.862; 95% CI, 1.22 to 2.72; P = .0017; c-index, 0.673). In a Cox regression model, dichotomized ligand expression was significantly associated with progression-free survival (PFS) and overall survival (OS). EREG PFS hazard ratio (HR) was 0.41 (95% CI, 0.274 to 0.609; P < .001; time-dependent c-index [Ctau index], 0.640), and AREG PFS HR was 0.43 (95% CI, 0.29 to 0.64; P < .001; Ctau index, 0.627). EREG OS HR was 0.42 (95% CI, 0.28 to 0.63; P < .0001; Ctau index, 0.639), and AREG OS HR was 0.40 (95% CI, 0.27 to 0.64; P < .0001; Ctau index, 0.625). There was no predictive power of ligand expression in patients with KRAS mutation. CONCLUSION: Expression of EGFR ligands in primary tumors significantly predicts outcome in KRAS WT cmCRC treated with cetuximab and irinotecan.

Predicting outcomes for children with neuroblastoma using a multigene-expression signature: a retrospective SIOPEN/COG/GPOH study

Abstract: Summary Background More accurate prognostic assessment of patients with neuroblastoma is required to better inform the choice of risk-related therapy. The aim of this study is to develop and validate a gene-expression signature to improve outcome prediction. Methods 59 genes were selected using an innovative data-mining strategy, and were profiled in the largest neuroblastoma patient series (n=579) to date using real-time quantitative PCR starting from only 20 ng of RNA. A multigene-expression signature was built using 30 training samples, tested on 313 test samples, and subsequently validated in a blind study on an independent set of 236 tumours. Findings The signature has a performance, sensitivity, and specificity of 85·4% (95% CI 77·7–93·2), 84·4% (66·5–94·1), and 86·5% (81·1–90·6), respectively, to predict patient outcome. Multivariate analysis indicates that the signature is a significant independent predictor of overall survival and progression-free survival after controlling for currently used risk factors: patients with high molecular risk have a higher risk of death from disease and higher risk of relapse or progression than patients with low molecular risk (odds ratio 19·32 [95% CI 6·50–57·43] and 3·96 [1·97–7·97] for overall survival and progression-free survival, respectively, both p MYCN status, age, International Neuroblastoma Staging System stage, ploidy, International Neuroblastoma Pathology Classification grade of differentiation, and mitosis karyorrhexis index (odds ratios between 4·81 and 10·53 depending on the model for overall survival and 3·68 [95% CI 2·01–6·71] for progression-free survival). Interpretation The 59-gene expression signature is an accurate predictor of outcome in patients with neuroblastoma. The signature is an independent risk predictor, identifying patients with an increased risk of poor outcome in the current clinical-risk groups. The method and signature is suitable for routine laboratory testing, and should be evaluated in prospective studies. Funding The Belgian Foundation Against Cancer, the Children Cancer Fund Ghent, the Belgian Society of Paediatric Haematology and Oncology, the Belgian Kid's Fund and the Fondation Nuovo-Soldati (JV), the Fund for Scientific Research Flanders (KDP, JH), the Fund for Scientific Research Flanders, the Institute for the Promotion of Innovation by Science and Technology in Flanders, Strategisch basisonderzoek, the Fondation Fournier Majoie pour l'Innovation, the Instituto Carlos III, the Italian Neuroblastoma Foundation, the European Community under the FP6, and the Belgian programme of Interuniversity Poles of Attraction.

RTPrimerDB: the portal for real-time PCR primers and probes

Abstract: RTPrimerDB (http://www.rtprimerdb.org) is a freely accessible database and analysis tool for real-time quantitative PCR assays. RTPrimerDB includes records with user submitted assays that are linked to genome information from reference databases and quality controlled using an in silico assay evaluation system. The primer evaluation tools intended to assess the specificity and to detect features that could negatively affect the amplification efficiency are combined into a pipeline to test custom-designed primer and probe sequences. An improved user feedback system guides users and submitters to enter practical remarks and details about experimental evaluation analyses. The database is linked with reference databases to allow the submission of assays for all genes and organisms officially registered in Entrez Gene and RefSeq. Records in RTPrimerDB are assigned unique and stable identifiers. The content is provided via an interactive web-based search system and is available for download in the recently developed RDML format and as bulk export file. RTPrimerDB is a one-stop portal for high-quality and highly annotated real-time PCR assays.

SURVEY AND SUMMARY RDML: structured language and reporting guidelines for real-time quantitative PCR data

Abstract: The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.

RDML: structured language and reporting guidelines for real-time quantitative PCR data

Abstract: The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.

Widespread Dysregulation of MiRNAs by MYCN Amplification and Chromosomal Imbalances in Neuroblastoma: Association of miRNA Expression with Survival

Abstract: 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.

Antitumor activity of the selective MDM2 antagonist nutlin-3 against chemoresistant neuroblastoma with wild-type p53.

Abstract: Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas.The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX(20)) or with mutant p53 (UKF-NB-3(r)VCR(10)). Activation of the p53 pathway was assessed by expression analysis of p53 target genes, flow cytometric cell cycle analysis, and apoptosis assays. Mice with established chemoresistant tumor xenografts were treated orally with nutlin-3 or vehicle control (n = 5-10 mice per group) and were used to evaluate effects on tumor growth, p53 pathway activity, and metastatic tumor burden. All statistical tests were two-sided.Nutlin-3 induced a similar activation of the p53 pathway in UKF-NB-3 and UKF-NB-3(r)DOX(20) cells, as evidenced by increased expression of p53 target genes, G(1) cell cycle arrest, and induction of apoptosis. No such response was observed in UKF-NB-3(r)VCR(10) cells with mutant p53. Oral administration of nutlin-3 to UKF-NB-3(r)DOX(20) xenograft-bearing mice led to inhibition of primary tumor growth (mean tumor volume after 3 weeks of treatment, nutlin-3- vs vehicle-treated mice: 772 vs 1661 mm(3), difference = 890 mm(3), 95% confidence interval = 469 to 1311 mm(3), P < .001), p53 pathway activation, and reduction in the extent of metastatic disease. The growth of UKF-NB-3(r)VCR(10) xenografts was unaffected by nutlin-3.Nutlin-3 activates the p53 pathway and suppresses tumor growth in this model system of chemoresistant neuroblastoma, provided that wild-type p53 is present.

Comparison of miRNA profiles of microdissected Hodgkin/Reed-Sternberg cells and Hodgkin cell lines versus CD77+ B-cells reveals a distinct subset of differentially expressed miRNAs.

Abstract: Classical Hodgkin lymphoma (cHL) is characterized by the presence of malignant Hodgkin and Reed Sternberg (HRS) cells. The scarcity of tumour cells in lymphoma biopsies has hampered genetic analyses of HRS cells, including microRNA (miRNA) expression profiling. We determined the expression of 360 miRNAs in microdissected HRS cells from nine cHL patients. These miRNA profiles were compared to those from four cHL cell lines and CD77+ B-cells, yielding a distinct cHL signature of 12 over- and three underexpressed miRNAs. Our data suggest that miRNAs are implicated in the pathogenesis of Hodgkin lymphoma and prompt further investigations concerning their role in cHL.

Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts

Abstract: Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos. The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated. Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.

Meta-mining of neuroblastoma and neuroblast gene expression profiles reveals candidate therapeutic compounds.

Abstract: Purpose: Neuroblastoma is a heterogeneous childhood tumor with poor survival outcome for the aggressive type despite intensive multimodal therapies. In this study, we aimed to identify new treatment options for neuroblastoma based on integrative genomic analysis. Experimental Design: The Connectivity Map is a database comprising expression profiles in response to known therapeutic compounds. This renders it a useful tool in the search for potential therapeutic compounds based on comparison of gene expression profiles of diseased cells and a database of profiles in response to known therapeutic compounds. We have used this strategy in the search for new therapeutic molecules for neuroblastoma based on data of an integrative meta-analysis of gene copy number and expression profiles from 146 primary neuroblastoma tumors and normal fetal neuroblasts. Results: In a first step, a 132-gene classifier was established that discriminates three major genomic neuroblastoma subgroups, reflecting inherent differences in gene expression between these subgroups. Subsequently, we screened the Connectivity Map database using gene lists generated by comparing expression profiles of fetal adrenal neuroblasts and the genomic subgroups of neuroblastomas. A putative therapeutic effect was predicted for several compounds of which six were empirically tested. A significant reduction in cell viability was shown for five of these molecules: 17-allylamino-geldanamycin, monorden, fluphenazine, trichostatin, and rapamycin. Conclusions: This proof-of-principle study indicates that an integrative genomic meta-analysis approach with inclusion of neuroblast data enables the identification of promising compounds for treatment of children with neuroblastoma. Further studies are warranted to explore in detail the therapeutic potential of these compounds.

RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts.

Abstract: The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR. The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification. Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.

The emerging molecular pathogenesis of neuroblastoma: implications for improved risk assessment and targeted therapy.

Abstract: 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.

An Assessment of Air As a Source of DNA Contamination Encountered When Performing PCR

Abstract: Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination.

External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data

Abstract: 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.

Unreliable Real-Time PCR Analysis of Human Endogenous Retrovirus-W (HERV-W) RNA Expression and DNA Copy Number in Multiple Sclerosis

Abstract: Editor: The potential role of human endogenous retroviruses in the pathogenesis of multiple sclerosis (MS) has been the subject of many studies since the discovery of MSRV, founder member of the HERV-W family. Two such studies from Prof. Power’s group in Canada, recently published in AIDS Research and Human Retroviruses, give us serious cause for concern. The findings of both studies were based on the use of real-time polymerase chain reaction (PCR) assays for the quantification of HERV-W RNA and DNA levels in brain, blood, and cerebrospinal fluid samples from patients and controls. We consider that technical flaws in the real-time PCRs employed in these studies are of such a severe and fundamental nature that the assays are unable to generate accurate or reliable data and that the conclusions of the papers are therefore unlikely to be valid. One of the critical parameters used to assess the quality of real-time PCR assays is the slope of the regression line derived from 10-fold dilutions of calibration standards. Ideally, in a perfect PCR with 100% efficiency, the slope should be 3.32 (21⁄4 10), but for practical purposes slopes within a range from 3.10 to 3.59 are generally regarded as acceptable. This represents an acceptable efficiency range of 90–110%. In view of this, we were disturbed to see slope values of 1.365 for syncytin-1 DNA and 2.276 for GAPDH DNA presented in the legend to Fig. 1, and 1.857 for syncytin-1 RNA in the legend to Fig. 2 of the paper by Antony et al. (NB: Syncytin-1 is a member of the HERV-W family and GAPDH is a reference gene.) Applying the equation, E1⁄4 10 1 to the syncytin-1 DNA slope value of 1.365 gives an apparent PCR efficiency (E) of 4.4, i.e., 440%, which is entirely implausible since Taq polymerase cannot produce more than a doubling of the number of DNA molecules with each PCR cycle. An efficiency of 440% would imply a 5-fold increase in DNA with each PCR cycle, which is impossible on theoretical grounds. Implausibly high apparent PCR efficiencies are a well-recognized problem, especially with SYBR Green I quantitative real-time assays, and may be due to the generation of primer dimers or spurious amplification products yielding excessive fluorescent signal, systematic pipetting errors, or to dilution of reverse transcriptase polymerase chain reaction (RT-PCR) inhibitors with increasing template dilution. A quantitative PCR assay with such an extremely aberrant regression line slope value would not be expected to be capable of yielding accurate or reliable data suitable for publication. Evidence of unreliable quantification resulting from the use of such defective PCR assays is seen in Fig. 1d of the same paper. Figure 1d shows GAPDH DNA copy numbers in brain-derived DNA extracted from multiple sclerosis patients and controls. Since GAPDH is a ‘‘single copy’’ gene we would expect a fixed number of GAPDH DNA copies per mg of extracted DNA (*3 10 copies=mg DNA). However, Fig. 1d does not show a fixed number of copies but an extraordinarily wide spread of copy numbers that extends over a 100-fold range. Even if we take into account the presence of a number of GAPDH pseudogenes that might be detected by the PCR assay, the problem of extremely high variability remains because the number of pseudogenes would not be expected to vary significantly between different individuals. The more recent paper by Antony et al. uses some of the same PCR assays as those discussed above. The grossly aberrant slope value of 1.365 for the ERVWE1 (syncytin-1) PCR is the same in both papers (Table 2 of reference 4). Further evidence of the unreliability of the data generated by these assays is provided by Table 3, which presents the coefficients of variation (CV%) of the PCR assays. Table 3 indicates, for example, that the overall intraassay variation for ERVWE1 (non-MS brain) is 10.15% based on raw Ct values. The figure of 10.15% is not worrying per se until we consider that this is the figure derived from raw Ct values that are logarithmic in nature and therefore grossly underestimate the true degree of variation that would be evident if the Ct values were converted into linear values (i.e., copies=ml) prior to calculating the CV%. Schmittgen et al., referring to this matter, state that the ‘‘Presentation of statistical data calculated from the raw Ct values falsely represents the error and should be avoided.’’ A 10.15% raw Ct figure for ERVWE1 would give an unacceptably large CV% (>100%) upon conversion to linear values. Reliable and accurate quantification cannot be achieved by assays with such high coefficients of variation. In addition to these major failings, we note that neither publication assesses RNA integrity and purity, which are critical parameters for evaluating the validity of RNA quantification by real-time PCR. Furthermore, correct usage of the 2 method, applied in both papers, requires evidence that PCR efficiencies for targets used for relative quantification are equal. The authors’ data clearly show that they are not equal, hence this method is inappropriate. AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 25, Number 3, 2009 a Mary Ann Liebert, Inc. DOI: 10.1089=aid.2008.0270

Standardization of qPCR and RT-qPCR

Abstract: T he perceived ease of use of real-time quantitative PCR (qPCR) and reverse transcription PCR (RT-qPCR) technology has revolutionized life science research. Its effectiveness at amplification and quantification of low levels of nucleic acids has driven the emergence of numerous applications, including cellular mRNA and miRNA quantification, biomarker discovery and validation, microbial quantification, cancer risk assessment, gene dosage determination, and detection of extremely low copy targets for forensic investigations. This, in turn, has resulted in an abundance of publications utilizing qPCR data obtained with diverse reagents, protocols, analysis methods, and reporting formats. Unfortunately, few papers report in detail how these results were obtained. This lack of clarity and transparency has led to concern in the research community over the reliability of qPCR data interpretation and the real danger of the scientific literature being corrupted with publications reporting erroneous and conflicting results. This has already occurred in some cases, resulting, for example, in retraction of a Science “Breakthrough of the Year 2005” report1. Now that qPCR has come of age, standardization is needed to ensure its validity, prompting the recent formulation of guidelines to increase experimental transparency, promote consistency between laboratoReprint from

Correlation between porcine PPARGC1A mRNA expression and its downstream target genes in backfat and longissimus dorsi muscle.

Abstract: Knowledge of in vivo relationship between the coactivatorPPARGC1A and its target genes is very limited, especially in the pig. In this study, a real-time PCR experiment was performed onlongissimus dorsi muscle (MLD) and backfat with 10 presumedPPARGC1A downstream target genes, involved in energy and fat metabolism, to identify possible relationships withPPARGC1A mRNA expression in vivo in the pig (n = 20). Except forUCP3 andLPL, a very significant difference in expression was found between MLD and backfat for all genes (P < 0.01). Hierarchical cluster analysis and the significant pairing of mRNA expression data between sampling locations suggested a genetic regulation of the expression of several target genes. A positive correlation withPPARGC1A was found forCPT1B, GLUT4, PDK4, andTFAM (P < 0.0001). A negative correlation was found forUCP2, FABP4, LEP (P < 0.0001), andTNF (P = 0.0071). No significant correlation was detected forUCP3 andLPL. This study provides evidence for a clear difference in mRNA expression of crucial genes in fat and energy metabolism between 2 important tissues. Our data suggest a clear impact ofPPARGC1A on energy and lipid metabolism in vivo in the pig, through several of these downstream target genes.

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.

Microrna signatures in genetic subtypes of t-cell acute lymphoblastic leukemia

Abstract: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that accounts for about 15 percent of ALL cases. Leukemic transformation of immature thymocytes is caused by a multistep pathogenesis involving numerous genetic abnormalities providing uncontrolled cell growth. Accumulating evidence suggests the presence of at least 5 different molecular-cytogenetic subgroups in T-ALL, ie. TAL/LMO , TLX1 , TLX3 , HOXA and MYB . Recently, non coding microRNAs were discovered as important regulators of gene and/or protein expression and subsequently shown to be directly implicated in cancer. Nevertheless, it is currently unclear in which way deregulated miRNA expression may contribute to the pathogenesis of T-cell acute leukemia. In this study, we investigated whether different genetic subgroups in T-ALL are characterized by distinct miRNA expression patterns. Therefore, we profiled a total of 360 miRNAs through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR in a genetically well characterized T-ALL patient cohort (n=52), including 11 HOXA (3 MLL rearranged, 5 inv(7)(p15q35) and 3 CALM - AF10 ), 16 TAL/LMO (7 LMO2 rearranged, 8 TAL1 rearranged, 1 LMO2/TAL1 rearranged), 11 TLX3 and 5 TLX1 rearranged patient samples. Since T-ALL blasts originate from maturating T lymphocytes, we also profiled different subsets of sorted T-cell populations (CD34 + , CD4 + /CD8 + /CD3 − , CD4 + /CD8 + /CD3 + , CD4 + SP and CD8 + SP). These miRNA profiles of normal T-cells served as a negative control for the identification of deregulated miRNA expression that may be truly leukemia associated. SAM analysis (t-test and wilcoxon, FDR=0) identified significant and differentially expressed miRNAs between the HOXA, TLX3 and TAL/LMO subgroups. No significant and differentially expressed miRNAs were obtained for the TLX1 subgroup, probably due to the limited number of patient samples. The HOXA subgroup showed specific up-regulation of miR-196a and miR-196b , which are encoded at the HOXB and HOXA cluster, respectively, but no significantly down-regulated miRNAs could be identified in this subgroup. The TLX3 subgroup was uniquely characterized by the up-regulation of miR-99a , miR-125b , let-7c , miR-508 and miR-509 , and down-regulation of miR-127 and miR-182 . Finally, specific up-regulation of miR-424 , miR-148a , miR-422 , miR-362 , miR-148a , miR-502 , miR-10a , miR-200c , miR-31 , miR-660 and miR-15b , was identified in the TAL/LMO rearranged subgroup, which was also characterized by the specific down-regulation of miR-99b , miR-155 , miR-125a , miR-153 , miR-135a , miR-34a and miR-193b . Next, we evaluated the expression pattern of all significant and differentially expressed miRNAs in the different subsets of sorted T-cell populations. The expression patterns of these miRNAs could be classified into consistently active, completely absent or temporally regulated during T-cell development. For the miRNAs showing a temporal regulation during T-cell maturation, their differential expression in T-ALL subtypes may reflect differences in the maturation arrest of the T-cell of origin, rather than pointing to an oncogenic event. Nevertheless, their constitutive (in)activation in primary T-ALL patients could still be of oncogenic relevance, similar to transcription factors like TAL1 or LMO2 which also show a temporal regulation during T-cell maturation. In contrast, some other miRNAs showed no expression in any of the T-cell populations, providing stronger evidence that their activation in specific T-ALL subtypes may contribute to T-ALL pathogenesis. In conclusion, this study shows that molecular-cytogenetic subgroups in T-ALL are characterized by a specific miRNA expression signature. In addition, correlation of our findings to the expression of these miRNAs in normal T-cell subsets may guide us to the miRNAs with true oncogenic potential. This report paves the way for further investigation directed at the role of these miRNAs in the pathogenesis of T-ALL, which may provide us with further insight in the oncogenic pathways that are (in)activated in different T-ALL subgroups. Ultimately, these deregulated miRNAs may offer new targets for therapeutic intervention.

New Guidelines Seek to Promote Accurate Interpretation of Data and Reliable Results Standardization of qPCR and RT- qPCR

Abstract: T he perceived ease of use of real-time quantitative PCR (qPCR) and reverse transcription PCR (RT-qPCR) technology has revolutionized life science research. Its effectiveness at amplification and quantification of low levels of nucleic acids has driven the emergence of numerous applications, including cellular mRNA and miRNA quantification, biomarker discovery and validation, microbial quantification, cancer risk assessment, gene dosage determination, and detection of extremely low copy targets for forensic investigations. This, in turn, has resulted in an abundance of publications utilizing qPCR data obtained with diverse reagents, protocols, analysis methods, and reporting formats. Unfortunately, few papers report in detail how these results were obtained. This lack of clarity and transparency has led to concern in the research community over the reliability of qPCR data interpretation and the real danger of the scientific literature being corrupted with publications reporting erroneous and conflicting results. This has already occurred in some cases, resulting, for example, in retraction of a Science “Breakthrough of the Year 2005” report1. Now that qPCR has come of age, standardization is needed to ensure its validity, prompting the recent formulation of guidelines to increase experimental transparency, promote consistency between laboratoReprint from

Chromosome 3p microsatellite allelotyping in neuroblastoma: a report on the technical hurdles

Abstract: Pinpointing critical regions of recurrent loss may help localize tumor suppressor genes. To determine the regions of loss on chromosome 3p in neuroblastoma, we performed loss of heterozygosity analysis using 16 microsatellite markers in a series of 65 primary tumors and 29 neuroblastoma cell lines. In this study, we report the results and discuss the technical hurdles that we encountered during data generation and interpretation that are of relevance for current studies or tests employing microsatellites. To provide functional support for the implication of 3p tumor suppressor genes in this childhood malignancy, we performed a microcell-mediated chromosome 3 transfer in neuroblastoma cells.