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Journal Article

New primer strategy improves precision of differential display

01 May 1995-BioTechniques (Biotechniques)-Vol. 18, Iss: 5, pp 842-850
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.
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Journal ArticleDOI
09 Jan 1998-Cell
TL;DR: The expression pattern of NAP and the phenotypes caused by its misexpression suggest that it functions in the transition between growth by cell division and cell expansion in stamens and petals.

555 citations


Cites methods from "New primer strategy improves precis..."

  • ...(Zhao et al., 1995), except that 33P-dATP (Amersham) was used, high-stringency annealing was at 558C, and arbitrary primers were References LH-AP (59TGCCGAAGCTTN7; N7 was seven arbitrary bases, which for the primers in Figure 3A were LH-AP4, 59CTGAGTG39 and LH- Aida, M., Ishida, T., Fukaki, H.,…...

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Journal ArticleDOI
TL;DR: Novel components of a complex, coordinated genetic program that is induced in striatal cells in response to various dopaminergic manipulations are identified, including CREM, CHOP, and MAP kinase phosphatase-1.
Abstract: Dopamine acting in the striatum is necessary for normal movement and motivation. Drugs that change striatal dopamine neurotransmission can have long-term effects on striatal physiology and behavior; these effects are thought to involve alterations in gene expression. Using the 6-hydroxydopamine lesion model of Parkinson's disease and differential display PCR, we have identified a set of more than 30 genes whose expression rapidly increases in response to stimulation of striatal dopamine D1 receptors. The induced mRNAs include both novel and previously described genes, with diverse time courses of expression. Some genes are expressed at near-maximal levels within 30 min, whereas others show no substantial induction until 2 hr or more after stimulation. Some of the induced genes, such as CREM, CHOP, and MAP kinase phosphatase-1, may be components of a homeostatic response to excessive stimulation. Others may be part of a genetic program involved in cellular and synaptic plasticity. A very similar set of genes is induced in unlesioned animals by administration of the psychostimulant cocaine or the antipsychotic eticlopride, although in distinct striatal cell populations. In contrast to some previously described early genes, most of the novel genes are not induced in cortex by apomorphine, indicating specificity of induction. Thus we have identified novel components of a complex, coordinated genetic program that is induced in striatal cells in response to various dopaminergic manipulations.

334 citations

Journal ArticleDOI
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

Journal ArticleDOI
TL;DR: Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation.
Abstract: Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx-1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx-2, which is found predominantly in proliferating osteoblasts. S...

292 citations

Journal ArticleDOI
TL;DR: There was no correlation between concentration of detectable TDF and the fragment size, stressing the high stringency of the amplification reactions, and the detection level of the cDNA-AFLP method was determined using TDFs of various sizes and composition.
Abstract: The method of cDNA-AFLP allows detection of differentially expressed transcripts using PCR This report provides a detailed and updated protocol for the cDNA-AFLP procedure and an analysis of interactions between its various parameters We studied the effects of PCR cycle number and template dilution level on the number of transcript derived fragments (TDFs) We also examined the use of magnetic beads to synthesise cDNA and the effect of MgCl2 concentration during amplification Finally, we determined the detection level of the cDNA-AFLP method using TDFs of various sizes and composition We could detect TDFs corresponding to a single copy per cell of a specific transcript in a cDNA-AFLP pattern, indicating high sensitivity of the method Also, there was no correlation between concentration of detectable TDF and the fragment size, stressing the high stringency of the amplification reactions Theoretical considerations and specific applications of the method are discussed

257 citations


Cites methods from "New primer strategy improves precis..."

  • ...Various modifications have been attempted, such as improvement of primer design (Zhao et al., 1995; Diachenko et al., 1996) and the combination of various methods with RNA-fingerprinting (Ivanova and Belyavsky, 1995; Kato, 1996)....

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