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.
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TL;DR: A novel molecular mechanism of cell cycle control under nitrosative stress is proposed based on the experimental results and bioinformatics analysis of the fission yeast Schizosaccharomyces pombe.
23 citations
TL;DR: A non-radioisotopic method to display differentially expressed cDNA bands is developed and a misjudged band excision wastes days of effort and causes frustration in the subsequent Northern hybridization.
Abstract: Differential display is a breakthrough in gene isolation methods (1,2). Compared with other gene isolation methods, it is powerful and relatively rapid. The method achieves isolation of differentially expressed genes by using four 3 ′-end degenerate primers and 20–40 5 ′-end arbitrary primers to perform RT–PCR. The PCR products are separated on a DNA sequencing gel and the differences between two or more cell lines are visualized and compared on an X-ray film. To isolate the differential bands from other closely spaced bands on a DNA sequencing gel, one must hold the X-ray film in one hand and cut the bands invisible to the eyes from a transparent gel or filter paper using the X-ray film as a reference. A misjudged band excision wastes days of effort and causes frustration in the subsequent Northern hybridization. To avoid unnecessary frustration and health hazardous radioisotopes, we have developed a non-radioisotopic method to display differentially expressed cDNA bands. To illustrate the method, a series of human lung adenocarcinoma cell lines (CL1, CLl-2 and CL1-5) with different invasive capabilities are used. Cells are collected by centrifuging at 300 g for 10 min and total RNA is extracted by using RNAzol B (Biotecx Laboratories, TX). The mRNA differential display method is performed essentially as described by Liang et al. (2) but with some modifications. Five μg of total RNA extracted from cell lines are incubated at 65 C for 15 min before reverse transcription is carried out. 100 pmol of digoxigenin (DIG) conjugated poly-T degenerate primers, e.g., DIG-T 12VG, are reverse transcribed in the presence of 0.5 mM dNTP, 10 mM DTT, 0.5 U/μl RNasin (GIBCO-BRL; Gaithersburg, MD), and 200 U of MMLV reverse transcriptase (GIBCO-BRL; Gaithersburg, MD). Reverse transcription is performed at 45 C for 60 min and is stopped by heating to 95 C for 5 min. The sample is then chilled on ice and diluted to 200 μl final volume. To amplify DNA, 2 μl of the 200 μl sample is subjected to a 40-cycle PCR amplification with the annealing temperature set at 38 C. After PCR amplification, samples are separated on a 6% SDS–polyacrylamide gel and transferred onto a nylon membrane using a semi-dry electro-blotting method. Before electro-blotting, the gel with one glass plate still attached is soaked in a transfer buffer (80 mM Tris–HCl, 120 mM borate, 2.5 mM EDTA, pH 8.3) for 30 min. The gel is then attached to a 3 mm filter paper and Figure 1. Non-radioisotopic differential display of three tumor cell lines with different invasive capabilities on a piece of nylon membrane. The primer sets used to generate the cDNA bands are shown on the top of the figure. The arrow heads indicate the differences between the three cell lines.
21 citations
TL;DR: This report describes a rapid, specific, nonradioactive fluorescent differential display methodology in which fluorescently differentially labeled anchored oligo(dT) downstream primers are used in the reaction, with subsequent analysis of fluorescently labeled PCR products on an automated sequencer.
Abstract: Differential display is a method for th e survey, analysis and comparison of gene e x pression in eukaryotic cells and tissues . Differential display involves isolation o f high-quality nondegraded RNA, selectiv e reverse transcription of polyadenylate d mRNA using specific anchored oligopol y deoxythymidine [oligo(dT)] primers, and the subsequent PCR amplification of th e cDNA with the same oligo(dT), an arbitrar y upstream primer and radioisotopes for la beling the PCR products. The radioisotop i cally labeled products are then separated on a sequencing gel. In this report, we d e scribe a rapid, specific, nonradioactive flu orescent differential display methodology in which fluorescently differentially labeled anchored oligo(dT) downstream primer s are used in the reaction, with subsequen t analysis of fluorescently labeled PCR prod ucts on an automated sequencer. Complet e gene expression profiles, containing mult i ple mRNA fingerprints are possible by th e simultaneous comparison of the multico l ored banding patterns of the fluorescentl y differentially labeled products from severa l primer combinations. This modification o f the differential display technique simplifie s the assay and increases the throughput o f high sample volumes required for compara tive gene expression studies in various clin ical applications .
18 citations
TL;DR: Differential display is used to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions to identify genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments.
6 citations