Differential Display Reverse Transcriptase Polymerase Chain Reaction Research Articles

Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: mechanical loading regulates osteopontin and myeloperoxidase.

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function.

Multiplex differential display identifies a novel zinc-finger protein repressed during withdrawal from cocaine

Multiplex differential display (MDD), a modification of differential display reverse transcriptase polymerase chain reaction (DD-PCR), was used to identify cocaine-dependent regulation of previously known and unknown gene products. Direct comparison of the MDD amplification profiles of duplicate, total RNA samples from the caudate putamen (CPu) of either vehicle or cocaine treated Sprague–Dawley rats indicated that the relative induction of a 240 bp (8G247) product, likely to represent c- fos mRNA, closely paralleled changes in c- fos mRNA as measured by Northern blot analysis. MDD and Northern blot analysis also revealed substantial repression of another PCR product (8G226) at 1 h and 1 day after repeated administration of cocaine. At 2 days after cocaine exposure, the level of 8G226 had returned to control levels. The DNA sequence of 8G226 exhibited near identity with a mouse zinc-finger protein (PZf) and is thus likely to represent a transcriptional regulator. Interestingly, the repression of 8G226 immediately after cocaine treatment is in direct contrast to the cocaine-dependent increase in expression documented for NGFI-A, another zinc-finger protein which also functions as a transcriptional regulator. Detailed characterization of the prolonged reduction in the expression of 8G226 may lead to the identification of additional regulatory pathways that produce changes in cellular response after repeated cocaine exposure.

Alterations in Gene Expression during Exposure of Bermudagrass to Low, Non-lethal Temperatures

Temperature is a limiting factor for plant growth. Warm-season turfgrasses can experience winter-kill when grown in the “transition zone.” On the other hand, when properly cold-acclimated, these same plants can withstand otherwise lethal temperatures. As part of our investigations into the biochemistry and molecular biology of cold acclimation in bermudagrass, total RNA from crowns (rhizome buds) isolated at different timepoints before and after chilling temperature exposure, was isolated by salt-buffer/phenol extraction, followed by LiCl precipitation and DNAse treatment. Differential display reverse transcriptase polymerase chain reaction (DD-RT-PCR) was performed using specific-(dT11NN) or variable-(dT12VN) anchor primers (where V = dA, dG and dC and N = dA, dG, dC or dT) for first strand cDNA synthesis by RT. The ss-cDNAs were converted to double stranded molecules and PCR amplified using a randomly chosen 10-mer primer paired with the same anchor primer used for cDNA synthesis. The dCTP32 labeled cDNAs were fractionated on non-denaturing polyacrylamide gels. Individual bands exhibiting differential expression between treated and nontreated samples were identified for reamplification, cloning, sequencing and further characterization of the differential nature of their expression by reverse northern hybridization and RT-PCR. Only those excised bands able to be reamplified using the anchor:10-mer pair were selected for cloning. To date, 90 variable-anchor:10-mer or specific-anchor:10-mer pairs have been screened. Of these, ≈27 have exhibited possible differential expression with one or more bands. Nucleotide (and deduced amino acid) sequence information was used to search on-line databases for similarity/homology with previously reported gene or protein sequences.

A simple method for isolation of PCR fragments from silver-stained polyacrylamide gels by scratching with a fine needle

▼PCR-based DNA and RNA fingerprinting techniques, including arbitrarily primed PCR (AP-PCR) and differential display reverse transcriptase PCR (ddRT-PCR), represent comprehensive genetic screening methods (Ref. 1). Recently, sensitive nonradioactive detection techniques have been introduced for visualization of fingerprints using silver staining of the PCR products after polyacrylamide gel electrophoresis (Ref. 2, 3). Characterization of polymorphic PCR fragments depends on prior isolation of the corresponding bands from stained and dried gels. In most protocols, PCR products of interest have been cut out of the dried gel complete with the paper support, and DNA eluted by boiling in elution buffer (Ref. 4). Reamplification of the eluted PCR fragments has been successful in up to 75% of the experiments. However, additional bands neighbouring the fragment of interest were frequently coamplified. To avoid coamplification of contaminating bands, the whole procedure of cutting, boiling and reamplification was repeated up to five times by some protocols (Ref. 5). Here we demonstrate simple and rapid reamplification of PCR products after isolation by gently scratching over the silver-stained band using a sterile disposable needle [Microlance 3 (Becton-Dickinson; 0.7 mm)] prewetted with PCR mastermix, without destroying the gel. After scratching the band from the gel, the needle is placed into 20 μl standard PCR reaction mix for 1 min and then discarded. The scratched PCR products are reamplified by 30 PCR cycles (Fig. 1). Using this scratching technique, we are able to reamplify PCR products of interest in more than 90% of the experiments. In addition to the simplicity and speed of

Reproducibility of nonradioactive DDRT-PCR method for detection of gene expression in squamous epithelial carcinoma cells of the upper aerodigestive tract

The analysis of the gene spectrum in tumor and normal cells may provide information about genes involved in the differentiation or in the genesis of tumors. Differential Display Reverse Transcriptase Polymerase Chain Reaction (DDRT-PCR) is an innovative method that enables quick analysis of almost all mRNA molecules expressed in the cells. In the trials, the mRNAs of keratinocytes and tumor cells were amplified by a number of oligonucleotide pairs after reverse transcription and resolved on polyacrylamide gel. A modified protocol for the amplification of cDNA probes was devised for detecting PCR products with silver nitrate. After reverse transcription of mRNAs, all cDNA samples were successfully amplified using the protocol devised for the silver nitrate DDRT-PCR, and the differentially expressed fragments were reproducibly demonstrated. The high reproducibility and feasibility of silver nitrate DDRT-PCR expand the alternatives for analyzing gene expression and detecting selectively expressed genes in different kinds of cells.

Differential display-mediated isolation of a genomic sequence for a putative mitochondrial LMW HSP specifically expressed in condition of induced thermotolerance in Arabidopsis thaliana (L.) heynh.

Plants of Arabidopsis thaliana pre-treated at 37 degrees C for 2 h can survive an otherwise lethal heat shock at 45 degrees C. Differential display reverse transcriptase-PCR (DDRT-PCR) was utilized to clone DNA fragments corresponding to mRNAs specifically expressed in conditions of induced thermotolerance or of expression of thermotolerance. One of these DDRT-PCR fragments enabled the isolation of a genomic clone pAt1.3EX, containing the sequence Athsp23.5, the gene for a low-molecular-weight (LMW) heat shock protein (HSP), AtHSP23.5. Athsp23.5 is low- or single-copy in the Arabidopsis genome and its open reading frame is interrupted by a 137 bp intron. Analysis of the sequence suggests AtHSP23.5 is targeted to the mitochondrion. The steady-state level of the AtHSP23.5 mRNA varied significantly according to the heat treatment, increasing on heat shock (transfer from 22 degrees C to 37 degrees C), with a further increase during expression of thermotolerance (transfer from 22 degrees C to 37 degrees C and then to 45 degrees C). Expression was low after an abrupt stress (from 22 degrees C to 45 degrees C). This behaviour was different from that observed for other LMW HSP mRNAs that were present at high level at 37 degrees C, but did not increase significantly in condition of expression of thermotolerance, and reached a considerable steady-state level also during the abrupt stress at 45 degrees C. The retrotranscription of AtHSP23.5 mRNA followed by amplification with two primers encompassing the intron allowed for the isolation of an almost full-length cDNA sequence. The sequence analysis of the two cDNAs obtained from condition 22 degrees C-->37 degrees C and condition 22 degrees C-->45 degrees C suggested that in both cases the intron had been correctly spliced. The importance of correct intron splicing in survival at high temperatures and the role of mitochondrial HSP in induction and expression of thermotolerance are discussed.

Molecular Cloning of a Novel mRNA Highly Expressed in Haemochromatotic Human Liver and Proliferating Cells

Pathological changes arising in altered cell biology and diseases such as cancer are driven by changes in gene expression. In the inherited disease haemochromatotis (HC) progressive iron loading of the parenchymal cells (hepatocytes) of the liver leads to cellular toxicity. If left untreated, fibrosis, cirrhosis and ultimately liver cancer occur. By using differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) techniques, we have identified and isolated several differentially displayed mRNAs that are excessively expressed or repressed in HC liver compared to normal human liver. One of these mRNAs was found to be strongly expressed in the liver of a patient with HC and in tumour tissue from a subject with hepatocellular carcinoma complicating HC (HC/HCC). The message of this gene was detected at a very low level in normal human liver. Northern analysis showed that this gene is also expressed in lymphocytes of HC patients and in MOLT-4 human T-lymphoid cells irrespective of iron status. The partial 1.0 kb cDNA sequence of the 9.5 kb transcript of this gene is unique and we propose that this gene may be related to cell proliferation and HC/HCC human liver.

Pulmonary TNFα is a critical mediator in Adult Respiratory Distress Syndrome

The development of effective pharmacotherapies to combat the Adult Respiratory Distress Syndrome (ARDS) is critically dependent upon: (1) the development of clinically-relevant animal models; (2) identification of inflammatory mediators centrally involved in eliciting lung injury; (3) understanding the inter-relationships or 'cross-talk' between pro and anti-inflammatory mediators which modulate the lung inflammation; and (4) the application of molecular techniques to isolate potentially novel genes involved in the development of ARDS. In this paper, we will present evidence from a rat model of microvascular lung injury produced by interleukin-2 (IL-2), that pulmonary TNFα is a primary and pivotal mediator of lung injury and that different modes of TNFα inhibition may represent feasible strategies to prevent ARDS. Furthermore, we will describe how the application of Differential Display Reverse Transcriptase Polymerase Chain Reaction (DDRT-PCR) can allow the rapid isolation of partial fragments of potentially new genes involved in ARDS. The products of such genes could represent future target sites for pharmacotherapeutic intervention.

Isolation of differentially expressed sequence tags from steroid-responsive cells using mRNA differential display

Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid-regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display reverse transcriptase PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3′ end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.