Caifu Chen - Academia.edu (original) (raw)

Papers by Caifu Chen

Journal of Biomolecular Techniques Jbt, 2006

We developed the snPlex Genotyping system to address the need for accurate genotyping data, high ... more We developed the snPlex Genotyping system to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. it is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The snPlex Genotyping system offers a high degree of flexibility and scalability, allowing the selection of customdefined sets of snPs for medium-to high-throughput genotyping projects. it is therefore suitable for a broad range of study designs. in this article we describe the principle and applications of the snPlex Genotyping system, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 snPs simultaneously. We present performance data from this control pool obtained by testing genomic dna samples from 44 individuals. in addition, we present data from a study that analyzed 521 snPs in 92 individuals. combined, both studies show the snPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by Taqman probe-based assays. The snPlex Genotyping system is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.

Journal of biomolecular techniques : JBT, 2005

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high ... more We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe s...

Methods in Molecular Biology, 2011

Quantification of the microRNAs (miRNAs) in cells or tissues is a crucial step in understanding t... more 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.

Nature Methods, 2014

nAture methods | ADVANCE ONLINE PUBLICATION | micrornAs are important negative regulators of prot... more nAture methods | ADVANCE ONLINE PUBLICATION | 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 transcriptionquantitative PCr (rt-qPCr) and (microarray) hybridization. in this study, we systematically compared 2 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. AnAlysis opportunity to provide platform-specific comments (Supplementary Note 13).

Methods in Molecular Biology, 2010

MicroRNAs (miRNAs) are ∼22 nucleotide regulatory RNA molecules that play important roles in contr... more 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.

Proceedings of the National Academy of Sciences, 1999

In this report, we present structural data for the murine Xist gene. The data presented in this p... more In this report, we present structural data for the murine Xist gene. The data presented in this paper demonstrate that the murine Xist transcript is at least 17.4 kb, not 14.3 kb as previously reported. The new structure of the murine Xist gene described herein has seven exons, not six. Exon VII encodes an additional 3.1 kb of information at the 3 end. Exon VII contains seven possible sites for polyadenylation; four of these sites are located in the newly discovered 3 end. Consequently, it is possible that several distinct transcripts may be produced through differential polyadenylation of a primary transcript. Alternative use of polyadenylation signals could result in size changes for exon VII. Two major species of Xist are detectable by Northern analysis, consistent with differential polyadenylation. In this paper, we propose a model for the role of the Xist 3 end in the process of X-chromosome counting and choice during embryonic development.

Oligonucleotides, 2009

RNA interference (RNAi) is a mechanism in which the introduction of small interfering RNAs (siRNA... more 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 identifi cation and validation, and therapeutics. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery effi ciency 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 quantifi cation of endogenous microRNAs in cells, to fulfi ll these needs. The application protocols developed enable robust quantifi cation of siRNA, including chemically modifi ed siRNA molecules, in vitro and in vivo.

Nucleic Acids Research, 2011

While a growing body of evidence implicates regulatory miRNA modules in various aspects of human ... more 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.

Nucleic Acids Research, 2008

MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of... more 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 preamplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional preamplification 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.

Nucleic Acids Research, 2005

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by ... more A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

Nature Cell Biology, 2011

deficiency 1-5 . So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC... more deficiency 1-5 . So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1,3 , indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a 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 Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as 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 p53 targets that play an essential role in restraining somatic reprogramming. ; ref. 6). This reprogramming process is rooted in the remarkable cellular plasticity retained during differentiation. The process can be triggered by exogenous expression of a set of defined ESC-specific transcription factors, Pou5f1 (also known as Oct4), Sox2, Klf4 and c-Myc (refs 6-9), which constitute the core regulatory circuits controlling pluripotency and self-renewal. Enforced expression of these reprogramming factors

Mammalian Genome, 2000

We have extended our earlier work to show that individual 14-20mer peptide nucleic acid probes di... more We have extended our earlier work to show that individual 14-20mer peptide nucleic acid probes directed against interspersed ␣-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 ␣-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 ∼400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, ∼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.

Mammalian Genome, 2007

Defining the identity of embryonic stem (ES) cells in quantitative molecular terms is a prerequis... more 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 mESspecific 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.

Mammalian Genome, 2006

During different periods of mammalian development, global changes in gene expression occur. Devel... more During different periods of mammalian development, global changes in gene expression occur. Developmental changes in global gene expression have been modeled as a restrictive process. To test the restriction model of global changes in gene expression, we have used embryonic stem (ES) cells as a model system for the early mammalian embryo. ES cells are pluripotent cells that can contribute to all cellular lineages of the developing mammalian fetus and are derived from early embryonic cells. Using this model system, we have studied a new class of RNAs called microRNAs that have been identified and shown to play a role in the direct regulation of messenger RNAs. Here we report the expression signature for 248 microRNAs in 13 independent murine ES cells, embryoid bodies, and somatic tissues. The expression profile for 248 mouse microRNAs was determined for embryonic stem cells, embryoid bodies, mouse embryos, mature heart, lung, liver, kidney, and brain. Characteristic microRNA expression signatures were observed for each evaluated sample. When the characteristic microRNA signatures for developmentally ordered samples were compared, immature samples exhibited a less complex microRNA transcript profile than did mature samples. Our data support a progressive model of microRNA gene expression. Based on the progressive increase in complexity of micro-RNA expression, we hypothesize that the mammalian developmental program requires a temporal coupling of expression between microRNAs and messenger RNAs to enable the developmental potential observed in mammalian ontogeny.