Analysis of Reverse Transcribed mRNA Using PCR and Polyacrylamide Gel Electrophoresis.
TL;DR: A method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast is described.
Abstract: The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel. The method has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, re-amplification and cloning of the differentially expressed PCR products, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. The technique is also popularly known as Differential Display Reverse Transcription (DDRT-PCR). After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives." Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of biology, DDRT-PCR has become a very popular molecular technique during the past two decades.
References
More filters
Journal Article•
TL;DR: It is shown that the purified cDNAs remain contaminated with unrelated cDNA sequences that may lead to the artifactual isolation of false positives in the subsequent steps of the differential display method.
Abstract: The recently described mRNA differential display method provides an attractive tool for the isolation of genes showing regulated expression in a variety of systems. A key step in this technique consists of the isolation of PCR-synthesized radioactive cDNAs corresponding to differentially expressed mRNAs. Here, we show that the purified cDNAs remain contaminated with unrelated cDNA sequences that may lead to the artifactual isolation of false positives in the subsequent steps of the method. A powerful assay for the detection and elimination of this contaminating material, allowing the specific isolation of clones corresponding to the regulated genes identified by the differential display, is provided.
142 citations
TL;DR: The method is used to identify genes involved in normal osteoblastic differentiation and in Paget's disease of bone and it has been widely used to study normal differentiation and pathological processes in a number of systems.
Abstract: Gene expression profiling relies on mRNA extraction from defined cell systems, which in the case of pathological processes necessarily results in the use of small quantities of tissues, sometimes as little as a few cells. This obviates the use of many systems of gene expression profiling and is best carried out using cDNA amplified by poly(A) reverse transcription polymerase chain reaction, which is capable of generating material representative of all the expressed genes in samples as small as one cell. Analysis of this material using subtractive hybridization compares the genes expressed at different stages of a biological/pathological process allowing identification of the all the genes upregulated during the process. The identification of the genes present is not dependent on their prior description or on the choice of genes used in a screen and as such the method is ideal for identifying novel genes or unsuspected genes. We have used the method to identify genes involved in normal osteoblastic differentiation and in Paget's disease of bone and it has been widely used to study normal differentiation and pathological processes in a number of systems. The method, its applications and its relationship with the other methods of gene expression profiling are reviewed.
46 citations
TL;DR: Here the authors report the use of a new programmable DNA sequencer, called the GenomyxLR (Genomyx, Foster City, USA), which reduces the number of reverse transcription reactions needed, increased reproducibility, reduced the incidence of false positives and increased the overall efficiency of the method.
Abstract: become a widely used method for the rapid identification of differentially expressed genes in a variety of eukaryotic systems (4). In the past three years, technical modifications have reduced the redundancy of anchored primers, decreased the number of reverse transcription reactions needed, increased reproducibility, reduced the incidence of false positives and increased the overall efficiency of the method (5). The original method separates cDNAs on a denaturing polyacrylamide gel. However, conventional DNA sequencers sometimes do not provide adequate gel resolution, resulting in the inadvertent isolation of several cDNAs from what appears to be a single band. These cDNAs may represent independent cDNA fragments of the same or very similar molecular weight (2), separated strands of a unique doublestranded cDNA molecule with or without Taq DNA polymerase-mediated addition of 3′-terminal adenosine nucleotide (1), truncated polymerase chain reaction (PCR) products of a unique cDNA (unpublished data) and fragments representing a unique cDNA with multiple polyadenylation sites (3). Here we report the use of a new programmable DNA sequencer, called the GenomyxLR (Genomyx, Foster City,
29 citations
Journal Article•
25 citations
25 citations