Search

Purpose: More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples.Experimental Design: Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples.Results: The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples.Conclusions: In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material. Clin Cancer Res; 17(24); 7684–92. ©2011 AACR.

PDF file - 132K

MicroRNAs are small noncoding RNAs that function by regulating target gene expression posttranscriptionally. They play a critical role in developmental and physiologic processes and are implicated in the pathogenesis of several human diseases including cancer. We examined the expression profiles of 241 human microRNAs in normal tissues and the NCI-60 panel of human tumor-derived cell lines. To quantify microRNA expression, we employed a highly sensitive technique that uses stem-loop primers for reverse transcription followed by real-time PCR. Most microRNAs were expressed at lower levels in tumor-derived cell lines compared with the corresponding normal tissue. Agglomerative hierarchical clustering analysis of microRNA expression revealed four groups among the NCI-60 cell lines consisting of hematologic, colon, central nervous system, and melanoma tumor–derived cell lines clustered in a manner that reflected their tissue of origin. We identified specific subsets of microRNAs that provide candidate molecular signatures characteristic of the tumor-derived cell lines belonging to these four clusters. We also identified specific microRNA expression patterns that correlated with the proliferation indices of the NCI-60 cell lines, and we developed evidence for the identification of specific microRNAs as candidate oncogenes and tumor suppressor genes in different tumor types. Our results provide evidence that microRNA expression patterns may mark specific biological characteristics of tumors and/or mediate biological activities important for the pathobiology of malignant tumors. These findings call attention to the potential of microRNAs to provide etiologic insights as well as to serve as both diagnostic markers and therapeutic targets for many different tumor types. [Cancer Res 2007;67(6):2456–68]

Supplementary Table 1 from Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

Supplementary Table 2 from Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

Supplementary Figure 1 from Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

Supplementary Table 4 from Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

Supplementary Figure 1 Legend from Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

Supplementary Table 3 from Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

The large-scale wind power integration into power system brings great impact on the stability of the grid voltage. In order to reduce the impact of wind power integration on the power system and improve the safety of wind power integration, it is necessary to carry out the research on reactive power compensation configuration of wind farm integration. Firstly, based on the actual situation of wind power project, study the principle of reactive power compensation configuration. Secondly, propose a theoretical calculation method of reactive power compensation configuration. On this basis, combined with the practical 300 MW wind farm project, analyze the reactive power compensation configuration example. Finally, through the modeling and simulation by PSASP software, the accuracy and effectiveness of the calculation method proposed by this paper are verified. The reactive power compensation configuration method studied in this paper is applicable to all wind farms connected to the power system and provides important support for voltage stability in the wind power integration project. It is of great significance to ensure the safe and stable operation of the power grid.

As a rotating equipment, the condenser can not only provide short-circuit capacity for the system, but also has good reactive power output characteristics. However, the longterm phase-leading operation of the condenser will affect the safety and stability of the condenser itself and the power grid. It is necessary to study the characteristics of the voltage regulation of the condenser in terms of reducing the transient over-voltage at the DC transmission end, dealing with the DC commutation failure, bipolar blocking and three-phase permanent fault of the AC system. Based on the analysis of the operation mechanism of the condenser and the case of the long-term leading-phase operation in ZLT, the operation status of the power grid near the ZLT converter station is simulated and analyzed by PSASP. The influence of different starting mode and DC power on the supporting ability of dynamic reactive power is calculated. The research shows that the operation mode of the condenser can meet the demand of system, which provides a theoretical support for the safety and stability of the system.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

We have developed a novel dual enzyme chemistry called rhAmp® SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci. Activation of the blocked primers occurs upon hybridization to its perfectly matched target, which eliminates or greatly reduces primer dimers. A thermostable hot-start RNase H2 cleaves the primer immediately 5’ of the ribose sugar, releasing the blocking group and allowing primer extension. PCR specificity is further improved with the use of a mutant Taq DNA polymerase, resulting in improved allelic discrimination. Signal generation is obtained using a universal reporter system which requires only two reporter probes for any bi-allelic SNP. 1000 randomly selected SNPs were chosen to validate the 95% design rate of the design pipeline. A subsampling of 130 human SNP targets was tested and achieved a 98% call rate, and 99% call accuracy. rhAmp SNP genotyping assays are compatible with various qPCR instruments including QuantStudioTM 7 Flex, CFX384TM, IntelliQube®, and Biomark HDTM. In comparison to TaqMan®, rhAmp SNP genotyping assays show higher signal (Rn) and greater cluster separation, resulting in more reliable SNP genotyping performance. The rhAmp SNP genotyping solution is suited for high-throughput SNP genotyping applications in humans and plants.

The large-scale wind power integration into power system brings great impact on the grid voltage stability. In order to reduce the influences of grid-connected wind power on the power system and improve the safety of wind power integration, it is necessary to conduct research on reactive power compensation configuration of wind farm integration. Firstly, based on the actual situation of wind power project, the principle of reactive power compensation configuration is studied. Secondly, a theoretical calculation method of reactive power compensation configuration is proposed. On this basis, combined with the practical 300MW wind farm project, the reactive power compensation configuration example is analyzed. Finally, through the modeling and simulation by power system analysis software package (PSASP), the accuracy and effectiveness of the calculation method proposed by this paper are verified. This paper put forward a reactive power compensation configuration method which is applicable to all wind farms connected to the power system. This provides important support for voltage stability in the wind power integration project.

Advances in high-throughput genotyping enable the generation of genome-scale data much more easily and at lower cost than ever before. However, small-scale and cost-effective high-throughput single-nucleotide polymorphism (SNP) genotyping technologies are still under development. In this study, we compared the performances of TaqMan, KASP and rhAmp SNP genotyping platforms in terms of their assay design flexibility, assay design success rate, allele call rate and quality, ease of experiment run and cost per sample. Fifty SNP markers linked to genes governing various agronomic traits of wheat were chosen to design SNP assays. Design success rates were 39/50, 49/50, and 49/50 for TaqMan, KASP, and rhAmp, respectively, and 30 SNP assays were manufactured for genotyping comparisons across the three platforms. rhAmp showed 97% of samples amplified while TaqMan and KASP showed 93% and 93.5% of amplifications, respectively. Allele call quality of rhAmp was 97%, while it was 98% for both TaqMan and KASP. rhAmp and KASP showed significantly better (p < 0.001) allele discrimination than TaqMan; however, TaqMan showed the most compact cluster. Based on the current market, rhAmp was the least expensive technology followed by KASP. In conclusion, rhAmp provides a reliable and cost-effective option for targeted genotyping and marker-assisted selection in crop genetic improvement.

Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher ScientificTM has recently commercialized the Applied BiosystemsTM QuantStudioTM 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.

SummaryExecution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a condition poised for lineage specification. This process is controlled at transcriptional, post-transcriptional and epigenetic levels and non-coding RNAs are contributors to this regulation complexity. Here we identify a molecular cascade initiated by a long non-coding RNA (lncRNA),Ephemeron(Epn), that modulates the dynamics of exit from naïve pluripotency.Epndeletion delays the extinction of ESC identity, an effect mediated by perduring expression of the pivotal transcription factor Nanog. In the absence ofEpn, Lin28a expression is reduced, resulting in an elevated level ofMirlet7gthat suppressesde novomethyltransferases Dnmt3a/b.Dnmt3a/bdeletion also retards exit from the ESC state, and is associated with delayed promoter methylation and slower down-regulation ofNanog.Altogether, our findings reveal a lncRNA/miRNA/DNA methylation axis that facilitates a timely stem cell state transition.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

INTRODUCTION Mouse zygotes and early blastomeres have totipotent cell fate potential, generating both embryonic and extraembryonic cell lineages during normal development. This totipotent potential is gradually restricted during development, with the first cell fate specification event being completed by the blastocyst stage. Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) exhibit a pluripotent potential similar to that of the epiblast in blastocysts, efficiently generating all embryonic cell types but rarely contributing to extraembryonic lineages in the placenta and yolk sac. Experimentally, ESCs and iPSCs can be induced into cells with expanded developmental potential, albeit with low efficiency. Such pluripotent stem cells are characterized by their bidirectional developmental potential (contributing to both embryonic and extraembryonic lineages) and their strong induction of the MuERV-L (MERVL) endogenous retroviruses (ERVs), both of which are features of totipotent two-cell (2C) blastomeres. The low efficiency in generating bipotential ESCs reflects the existence of multiple cellular and molecular impediments that restrict the pluripotent cell fate potential. RATIONALE We identified miR-34a microRNA (miRNA) as a potent regulator that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. miR-34a −/− pluripotent stem cells exhibit a bidirectional cell fate potential, generating both embryonic and extraembryonic cell lineages in multiple functional assays. Hence, the miR-34a −/− pluripotent stem cells provide a powerful experimental system to dissect the molecular mechanisms that restrict cell fate potential in ESCs and iPSCs. RESULTS miR-34a −/− ESCs and iPSCs exhibited an expanded cell fate potential, generating both embryonic and extraembryonic lineages in teratomas, embryoid bodies, and chimeric embryos. In particular, a single miR-34a −/− ESC injected into a recipient morula could yield progenies in both inner cell mass (ICM) and trophectoderm. Expression profiling studies comparing wild-type and miR-34a −/− pluripotent stem cells revealed a strong and specific induction of the MERVL ERVs, together with many MERVL-proximal genes, in miR-34a −/− ESCs and iPSCs. Whereas wild-type ESCs and iPSCs almost exclusively expressed Oct4, miR-34a −/− ESCs and iPSCs were heterogeneous, containing mutually exclusive populations with either Oct4 expression or MERVL induction. Because MERVL is a specific and highly expressed molecular marker for totipotent 2C blastomeres and for bipotential ESCs, we investigated the mechanism by which miR-34a regulates MERVL expression. We demonstrated that MERVL induction in miR-34a –deficient pluripotent stem cells is regulated at the transcriptional level, at least in part because of an increase of the transcription factor Gata2, a direct target of miR-34a . Knockdown of gata2 in miR-34a –deficient pluripotent stem cells phenocopied miR-34a overexpression, not only down-regulating the expression of MERVL but also abolishing their bipotential cell fate. Thus, miR-34a restricts cell fate potential and represses MERVL induction in pluripotent stem cells, at least in part through down-regulation of Gata2. CONCLUSION We have identified miR-34a as a noncoding RNA that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. The miR-34a / gata2 /MERVL axis plays an essential role in modulating the transition between pluripotent stem cells and bipotential stem cells in culture. Thus, an intricate network of protein-coding genes, noncoding RNAs, and endogenous retroviruses could act cooperatively to define cell fate plasticity and developmental potential in pluripotent stem cells.

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations. DOI: http://dx.doi.org/10.7554/eLife.23468.001

Totipotency is the ability of a single cell to form an entire embryo, including extraembryonic tissues, an ability we have yet to recapitalize, in vitro. In a recent paper published in Science, Choi et al. showed that pluripotent stem cells lacking microRNA miR-34a, have an expanded cell fate potential allowing differentiation into not only embryonic but also extraembryonic lineages.

Purpose: More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples. Experimental Design: Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples. Results: The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples. Conclusions: In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material. Clin Cancer Res; 17(24); 7684–92. ©2011 AACR.

Somatic reprogramming induced by defined transcription factors is a low efficiency process that is enhanced by p53 deficiency 1-5. To date, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1, 3, suggesting additional p53 targets may regulate this process. Here, we demonstrated that mir-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. mir-34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of mir-34a promoted iPSC generation without compromising self-renewal and differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three mir-34 miRNAs acted cooperatively in this process. Taken together, our findings identified mir-34 miRNAs as novel p53 targets that play an essential role in restraining somatic reprogramming.

MicroRNAs (miRNAs) regulate specific immune mechanisms, but their genome-wide regulation of T lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated, and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T lymphocytes) using locked nucleic acid inhibitors. Inhibition of these two highly upregulated miRNAs in CD4+ T cells was shown to increase proliferation by removing suppression of four target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T lymphocyte immunity, underlining the value of mapping global gene, protein, and miRNA expression.

Introduction: Molecular diagnostics and precise personalized care continue to increase the sensitivity and specificity requirements for detecting low (~5%) to ultra-low (4,000 false positives, resulting in a positive predictive value (PPV) of 90% detection sensitivity was achieved for genomic and cell-free DNA samples with 0 false positives, resulting in 100% PPV, a >40-fold error suppression. For FFPE DNA samples, the duplex adapters provided >30-fold PPV improvement compared to standard adapters. Conclusions: The duplex UMI adapters eliminate background NGS errors by collapsing duplicated reads using their unique tags. This leads to unprecedented accuracy in detecting true low-frequency variants, regardless of the input DNA source. Such advances are key to refining diagnostics and improving precision cancer care. Citation Format: Jiashi Wang, Kevin Lai, Madelyn Light, Layla Katiraee, Kristina Giorda, Mirna Jarosz, Yun Bao, Criss Walworth, David Kupec, Caifu Chen. Highly efficient duplex DNA tagging strategy improves accuracy of detecting ultra-low-frequency mutations through consensus read reconstruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 418.

Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and FFPE DNA. Furthermore, clinical samples are often available in limited quantity, making the detection of pathologic variants particularly challenging. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by “duplex sequencing”, where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. We present a novel library construction chemistry that eliminates adapter dimers and utilizes duplexed UMIs to increase sensitivity. The method employs a two-step ligation procedure that does not require size-selection or adjustment of AMPure bead ratios during cleanup. We achieve maximal library conversion using a unique, mutant DNA ligase and proprietary sequencing adapters that increase ligation efficiency and suppress chimera formation. We demonstrate performance using three sample types: sheared genomic DNA, cfDNA, and FFPE DNA, across a wide input range (1-1000 ng). To mimic performance with degraded DNA, we created libraries from mixtures of NA12878 and NA24385 cell line DNA sheared to 150 bp with inputs of 1-25 ng and mutant allele fractions (MAFs) down to 0.25%. Libraries were enriched using a 75 kb panel of xGen Lockdown Probes, followed by ultra-deep sequencing and variant calling. When compared to commercially available methods, our approach yielded a 50-100% increase in library complexity with significantly improved sensitivity to Citation Format: Ushati Das Chakravarty, Zac Zwirko, Yu Zheng, Madelyn Light, Kevin Lai, Keith Bryan, Scott Rose, Yun Bao, Mirna Jarosz, Caifu Chen. Detection of low-frequency variants from highly degraded DNA samples using a novel library preparation method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 430.

RNA interference (RNAi) is a mechanism in which the introduction of small interfering RNAs (siRNAs) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. Applications of RNAi include gene function and pathway analysis, target identification and validation, and therapeutics. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (posttransfection) and in animals (postinjection). We have leveraged the Applied Biosystems TaqMan®-based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. The application protocols developed enable robust quantification of siRNA, including chemically modified siRNA molecules, in vitro and in vivo.

The unique properties of embryonic stem cells (ESCs) to self-renew indefinitely or to differentiate to any cell type have great potential for clinical applications in regenerative medicine. MicroRNAs (miRNAs) are emerging as important regulators of post-transcriptional gene expression and have been implicated as crucial elements in regulating ESCs. Here, we review recent progresses in characterizing the role of miRNAs in the maintenance and development of ESCs.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurode- generative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

Defining the identity of embryonic stem (ES) cells in quantitative molecular terms is a prerequisite to understanding their functional characteristics. Little is known about the role of microRNAs (miRNAs) in the regulation of ES cell identity. Statistical analysis of miRNA expression revealed unique expression signatures that could definitively classify mouse ES (mES), embryoid bodies (mEB), and somatic tissues. Analysis of these data sets also provides further confirmation of the nonrestrictive expression of miRNAs during murine development. Using combined genome-wide expression analyses of both miRNAs and mRNAs, we observed both negative and positive correlations in gene expression between miRNAs and their predicted targets. ES-specific miRNAs were positively correlated with their predicted targets, suggesting that mES-specific miRNAs may have a different role or mechanism in regulating their targets in mES maintenance or differentiation. The concept of cellular identity has changed with technology; this study redefines cellular identity by a generic statistical method of known dimension.

A global decrease in microRNA (miRNA) levels is often observed in human cancers1,2, indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, we compared miRNA expression profiles of wild-type and p53-deficient cells. Here we describe a family of miRNAs, miR-34a–c, whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53 both in vitro and in vivo. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a programme of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation.

MicroRNAs are short (approximately 22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.

We have extended our earlier work to show that individual 14-20mer peptide nucleic acid probes directed against interspersed alpha-satellite sequences can specifically identify chromosomes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human genome by fluorescence in situ hybridization (FISH). The hybridization of a single PNA probe species directed against a highly abundant alpha-satellite DNA repeat sequence was sufficient to absolutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program "whole genome" approach based on approximately 400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, approximately 240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results demonstrate that chromosome-specific PNA probes are capable of detecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome "bar coding". Potential application of PNA probes for investigating repeat structure and function is also discussed.

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome.

Quantification of the microRNAs (miRNAs) in cells or tissues is a crucial step in understanding their biological functions. Development of the stem-loop reverse transcription procedure and TaqMan(®) miRNA assays enables accurate detection of miRNA expression levels by quantitative PCR. Increased experimental throughput permits the expression screening of larger number of miRNAs with small amounts of sample. Here, we demonstrate the use of both TaqMan(®) Array Card and OpenArray(®) platforms to accurately determine the level of miRNA gene expression in biological samples.

Quantification of the microRNAs (miRNAs) in cells or tissues is a crucial step in understanding their biological functions. Development of the stem-loop reverse transcription procedure and TaqMan(®) miRNA assays enables accurate detection of miRNA expression levels by quantitative PCR. Increased experimental throughput permits the expression screening of larger number of miRNAs with small amounts of sample. Here, we demonstrate the use of both TaqMan(®) Array Card and OpenArray(®) platforms to accurately determine the level of miRNA gene expression in biological samples.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

MicroRNAs (miRNAs) relevant to acute lymphoblastic leukemia (ALL) in children are hypothesized to be largely unknown as most miRNAs have been identified in non-leukemic tissues. In order to discover these miRNAs, we applied high-throughput sequencing to pooled fractions of leukemic cells obtained from 89 pediatric cases covering seven well-defined genetic types of ALL and normal hematopoietic cells. This resulted into 78 million small RNA reads representing 554 known, 28 novel and 431 candidate novel miR genes. In all, 153 known, 16 novel and 170 candidate novel mature miRNAs and miRNA-star strands were only expressed in ALL, whereas 140 known, 2 novel and 82 candidate novel mature miRNAs and miRNA-star strands were unique to normal hematopoietic cells. Stem-loop reverse transcriptase (RT)-quantitative PCR analyses confirmed the differential expression of selected mature miRNAs in ALL types and normal cells. Expression of 14 new miRNAs inversely correlated with expression of predicted target genes (-0.49 ≤ Spearman's correlation coefficients (Rs)≤ -0.27, P ≤ 0.05); among others, low levels of novel sol-miR-23 associated with high levels of its predicted (antiapoptotic) target BCL2 (B-cell lymphoma 2) in precursor B-ALL (Rs -0.36, P = 0.007). The identification of >1000 miR genes expressed in different types of ALL forms a comprehensive repository for further functional studies that address the role of miRNAs in the biology of ALL.

MicroRNAs (miRNAs) are ∼22 nucleotide regulatory RNA molecules that play important roles in controlling developmental and physiological processes in animals and plants. Measuring the level of miRNA expression is a critical step in methods that study the regulation of biological functions and that use miRNA profiles as diagnostic markers for cancer and other diseases. Even though the quantitation of these small miRNA molecules by RT-qPCR is challenging because of their short length and sequence similarity, a number of quantitative RT-qPCR-based miRNA quantitation methods have been introduced since 2004. The most commonly used methods are stem-loop reverse transcription (RT)-based TaqMan(®) MicroRNA assays and arrays. The high sensitivity and specificity, large dynamic range, and simple work flow of TaqMan(®) MicroRNA assays and arrays have made TaqMan analysis the method of choice for miRNA expression profiling and follow-up validation. Other methods such as poly (A) tailing-based and direct RT-based SYBR miRNA assays are also discussed in this chapter.