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: The 2-Delta Delta C(T) method as mentioned in this paper was proposed to analyze the relative changes in gene expression from real-time quantitative PCR experiments, and it has been shown to be useful in the analysis of realtime, quantitative PCR data.
139,407 citations
TL;DR: A method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction using a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer.
Abstract: Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
5,254 citations
TL;DR: Differential display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate differentially expressed genes and have been introduced to streamline the techniques.
295 citations
TL;DR: This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation and found that incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs.
Abstract: In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.
188 citations
Journal Article•
TL;DR: Modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods to increase the reproducibility and sensitivity of the mRNA differentialdisplay while still keeping the characteristics of the original method.
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.
154 citations