The Experts below are selected from a list of 31287 Experts worldwide ranked by ideXlab platform

A Fischer - One of the best experts on this subject based on the ideXlab platform.

Peng Liang - One of the best experts on this subject based on the ideXlab platform.

  • Proof-reading signal accuracy of gene expression by binary Differential Display
    Biotechnology Letters, 2010
    Co-Authors: Yong-jig Cho, Jonathan D. Meade, Blake R. Shester, Jamie C. Walden, Zhen Guo, Peng Liang
    Abstract:

    Differential Display (DD) is commonly used for identifying Differentially expressed genes. However, each cDNA species identified by DD must be verified so a “real difference” can be differentiated from false positives. Although Northern blot analysis is the gold standard it is labor intensive, time-consuming and requires a significant amount of RNA. To speed up and streamline the confirmation process, we developed a new strategy: binary Differential Display (BDD) based on the binding kinetics of the arbitrary primers in DD. After determining a cDNA sequence of interest from a DD screen, two more 13mer primers derived from the original arbitrary primer used can be designed to target a corresponding cDNA sequence of interest: one with perfect 5′-base matches and the other with additional mismatches at the 5′-base to the corresponding mRNA being confirmed. A separate reverse transcription and FDD are then performed with the same RNA samples being compared. BDD can quickly and accurately determine if a cDNA sequence identified by DD corresponds to a truly Differentially expressed gene. In addition, the method is especially useful when more than one cDNA sequence was recovered from a DD band where the masking effect of false-positives can be clearly resolved. Given its simplicity and limited RNA sample required, BDD can be used as a general strategy for rapid confirmation of Differentially expressed genes discovered by DD.

  • Differential Display analysis of gene expression in mammals: a p53 story.
    Cellular and molecular life sciences : CMLS, 2002
    Co-Authors: Susanne Stein, Peng Liang
    Abstract:

    Differential Display is used worldwide as a method to identify changes in gene expression and to discover novel genes that are involved in important biological pathways. The principle of Differential Display is the systematic amplification of the 3′ termini of messenger RNAs by using anchored oligo-dT primers in combination with upstream arbitrary primers. The separation of the polymerase chain reaction products by gel electrophoresis and their direct comparison allows the identification of Differentially regulated genes. Recently, fluorescent Differential Display was established as the first nonradioactive Differential Display system with equivalent sensitivity to originally 33P isotopic labeling method. Because of its simplicity, sensitivity, reproducibility and automation, which increase the throughput and accuracy, Differential Display has become one of the most widely used gene-screening methods in biomedical research involving mammals. This chapter provides a glimpse of the application of Differential Display in search of target genes of the p53 tumor suppressor gene.

  • A decade of Differential Display.
    BioTechniques, 2002
    Co-Authors: Peng Liang
    Abstract:

    It has been 10 years since the inventio n of Differential Display (DD), a conceptuall y simple methodology that allows the dete c tion and identification of Differentially e x pressed genes. In the past decade, the num ber of publications describing successfu l applications of DD has outnumbered thos e using any other competing methodologies , including subtractive hybridization, repr e sentational difference analysis, seria l analysis of gene expression, and DNA m i croarrays. This review will provide a glimpse of the current progress made in DD technological development, refinement, an d automation. Excellent examples of DD a p plications in studying a variety of biologica l problems, in such diverse biological sy s tems as bacteria, yeast, flies, plants, an d higher mammals, are presented to provide a roadmap for those who would like to pursu e a fruitful gene “fishing” expedition. Som e of the fundamental differences between DD and DNA microarrays are also discussed .

  • Detection of an mRNA polymorphism by Differential Display
    Molecular Biotechnology, 2001
    Co-Authors: Shan Liang, Peng Liang, S. Paul Rossby, Richard C. Shelton, D. Hal Manier, Amitabha Chakrabarti, Fridolin Sulser
    Abstract:

    Differential Display (DD) technology was utilized to compare programs of gene expression in primary cultures of human skin fibroblasts from normal volunteers and patients diagnosed with melancholic depression. Polymorphic transcripts of a single gene differing by one tandem repeat sequence of four nucleotides (TGAT) in the 3′ noncoding region were detected.

  • Multicolor fluorescent Differential Display.
    BioTechniques, 2001
    Co-Authors: Yong-jig Cho, Jonathan D. Meade, J.c. Walden, Xiangning Chen, Z. Guo, Peng Liang
    Abstract:

    Differential Display and DNA microarray have emerged as the two most popular methods for gene expression profiling. Here, we developed a multicolor fluorescent Differential Display (FDD) method that combines the virtues of both Differential Display in signal amplification and DNA microarray in signal analysis. As in DNA microarray, RNA samples being compared can be labeled with either a red or green fluorescent dye and Displayed in a single lane, allowing convenient scoring and quantification of the Differentially expressed messages. In addition, the multicolor FDD has a built-in signal proofreading capability that is achieved by labeling each RNA sample from a comparative study with both red and green fluorescent dyes followed by their reciprocal mixings in color. Thus, the multicolor FDD provides a platform upon which a sensitive and accurate gene expression profiling by Differential Display can be automated and digitally analyzed. It is envisioned that cDNAs generated by the multicolor FDD may also be used directly as probes for DNA microarray, allowing an integration of the two most widely used technologies for comprehensive analysis of gene expression.

Arthur B. Pardee - One of the best experts on this subject based on the ideXlab platform.

  • Principles of Differential Display.
    Methods in enzymology, 1999
    Co-Authors: Katherine J. Martin, Arthur B. Pardee
    Abstract:

    Publisher Summary The introduction of Differential Display (DD) and related techniques has contributed to the recent shift in focus from DNA genetics to expression genetics, especially in the field of cancer research. This chapter describes the principles of DD, including specific methods for working with the primers that have been used extensively. The chapter discusses the production of a DD gel, strategies for isolating and identifying DD band cDNAs, and information on performing high-throughput DD. DD is distinguished from related methods by a low stringency, competitive polymerase chain reaction (PCR) step that uses primer pairs to target the 3’ ends of messenger RNAs. One of the major criticisms of the DD technique has been the high number of false positives obtained in some studies.

  • Differential Display. A general protocol.
    Molecular biotechnology, 1998
    Co-Authors: Peng Liang, Arthur B. Pardee
    Abstract:

    Characterization of regulated gene expression in eukaryotic cells is essential for studying cell growth and differentiation as well as for understanding the molecular mechanisms of diseases. Differential Display was developed for such comparative studies by allowing a systematic and nonbiased screening for molecular differences at the level of mRNA expression between or among different cells or tissues. The essence of the method is to amplify messenger RNA 3' termini using a pair of anchored oligo-dT primer and a short primer with an arbitrary sequence. The amplified cDNAs labeled with radioisotope are then distributed on a denaturing polyacrylamide gel and visualized by autoradiography. Side-by-side comparison of mRNA species from two or more related samples allows identification of both up- and downregulation genes of interest. Some of the most recent improvements have been incorporated into this general protocol for Differential Display.

  • New primer strategy improves precision of Differential Display
    BioTechniques, 1995
    Co-Authors: Shanchuan Zhao, S. L. Ooi, Arthur B. Pardee
    Abstract:

    To increase the reproducibility and to reduce the false positives in the initial mRNA Differential Display, modified long composite primers were developed based on both mRNA Differential Display and RNA arbitrarily primed PCR fingerprinting methods. Ten-base nucleotides were added at the 5' ends of the primers used in the initial mRNA Differential Display. These included a restriction site to aid cloning. PCR began with one low-stringency cycle (40 degrees C for annealing) followed by 35 high-stringency cycles (60 degrees C for annealing). The modified method significantly improved the reproducibility and sensitivity of the mRNA Differential Display while still keeping the characteristics of the original method.

  • RECENT ADVANCES IN Differential Display
    Current opinion in immunology, 1995
    Co-Authors: Peng Liang, Arthur B. Pardee
    Abstract:

    Abstract Differential Display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate Differentially expressed genes. Methodological modifications have since been introduced to streamline the techniques. The major effor thas centered on how to eliminate false positives as approached from a variety of angles, ranging from RNA sample preparation, northern blot confirmation, primer length variation, to better experimental design.

  • [20] Analysis of altered gene expression by Differential Display
    Methods in enzymology, 1995
    Co-Authors: Peng Liang, David Bauer, Lidia Averboukh, Peter Warthoe, Markus Rohrwild, Heiko Muller, Mike Strauss, Arthur B. Pardee
    Abstract:

    Publisher Summary This chapter presents the analysis of altered gene expression by Differential Display. Temporal and spatial expression of the 100,000 different genes in a mammalian cell I must be highly regulated and tightly controlled as specificity determines life processes from the fertilized egg to death of the organism. The course of normal development as well as the pathological changes that arise in diseases such as cancer, whether caused by a single gene mutation or complex multigene effects, are driven by changes in gene expression. A fingerprinting technique for mRNAs would be very useful, for instance, by 2D electrophoresis, similar to 2D protein separation. A novel method named "Differential Display" (DDRT-PCR), according to polymerase chain reaction (PCR) terminology, was developed. This method provides a sensitive and flexible approach to the identification of genes that are Differentially expressed at the mRNA level. The method is directed toward the identification of Differentially expressed genes, detecting individual mRNA species that are changed in different sets of eukaryotic cells, and then permitting recovery and cloning of their DNA.

Eric D. Schwab - One of the best experts on this subject based on the ideXlab platform.

  • Modified Differential Display technique that eliminates radioactivity and decreases screening time.
    BioTechniques, 2000
    Co-Authors: Kenneth J. Pienta, Eric D. Schwab
    Abstract:

    Several techniques are available that detect variations in gene expression between cellular populations. These include subtractive hybridization (SH), Differential colony hybridization (DCH) and mRNA Differential Display, all based on the analysis of mRNA. The first two techniques, however, are limited because they require large amounts of mRNA for SH or several rounds of screening for DCH. Differential Display overcomes both of these limitations. However, the conventional Differential Display technique is plagued by false positives and is labor intensive. The identification of genes that are truly Differentially expressed, therefore, becomes a formidable task. We describe a modified Differential Display technique that overcomes the limitations of the conventional technique. This new technique eliminates a source of false positives, decreases the time required to screen a set of primers and reduces the use of radioactivity.

F.j. Livesey - One of the best experts on this subject based on the ideXlab platform.

  • Current Protocols in Human Genetics - Differential Display of mRNA by PCR
    Current Protocols in Human Genetics, 2001
    Co-Authors: F.j. Livesey, S P Hunt
    Abstract:

    This unit outlines the polymerase chain reaction (PCR)-based technique of mRNA Differential Display, which identifies genes that are Differentially expressed between cells or tissues. The basic protocol describes the actual Differential Display PCR reaction along with details of the identification, reamplification, and cloning of candidate Differentially expressed genes. A support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.

  • Differential Display of mRNA by PCR.
    Current protocols in human genetics, 2001
    Co-Authors: F.j. Livesey, S P Hunt
    Abstract:

    This unit outlines the polymerase chain reaction (PCR)-based technique of mRNA Differential Display, which identifies genes that are Differentially expressed between cells or tissues. The basic protocol describes the actual Differential Display PCR reaction along with details of the identification, reamplification, and cloning of candidate Differentially expressed genes. A support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.

  • Differential Display of mRNA by PCR.
    Current protocols in neuroscience, 1997
    Co-Authors: F.j. Livesey, C.p. Hunt
    Abstract:

    This unit outlines the polymerase chain reaction (PCR)-based technique of mRNA Differential Display, which identifies genes that are Differentially expressed between cells or tissues. The approach described here is a modification of the original method, referred to as single base-anchored Differential Display. The basic protocol describes the actual Differential Display PCR reaction along with details of the identification, reamplification, and cloning of candidate Differentially expressed genes. The support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.