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Cardiovascular Research 1997 35(3):414-421; doi:10.1016/S0008-6363(97)00169-7
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

The use of mRNA differential display for discovery of novel therapeutic targets in cardiovascular disease

Xinkang Wang and Giora Z Feuerstein*

Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA

* Corresponding author. Dept. of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA. Tel.: +1 (610) 270-6802; fax: +1 (610) 270-4114; e-mail: Giora_Z_Feuerstein@SBPHRD.COM@INET

Received 17 February 1997; accepted 23 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Critical procedure of...
 3 Applications of mRNA...
 4 Summary
 References
 
Recent advances in molecular biology techniques have provided powerful tools for discovery of novel genes relevant to both biological and pathological processes. mRNA differential display is an emerging technique for novel gene discovery and it has been successfully applied to many physiological and pathological conditions including normal development, cell differentiation, cancer, cardiovascular disease, inflammation and CNS disorders. In the present work, we briefly illustrate the critical procedure and highlight most recent technical improvements and modifications of this technology. Based upon the successful applications of this technique in cardiovascular research, it may provide a valuable and powerful investigational tool for the identification of novel therapeutic targets in cardiovascular diseases.

KEYWORDS Cardiovascular disease; Differential display; Gene expression; Therapeutic target


   1 Introduction
Top
 Abstract
 1 Introduction
2 Critical procedure of...
 3 Applications of mRNA...
 4 Summary
 References
 
Differential gene expression is essential for normal development and pathological processes. Therefore, discovery of differentially expressed genes is critical for the understanding of the molecular mechanisms involved in normal and pathological states, as well as providing new insights for discovery of novel molecular targets for pharmacological manipulation and gene therapy. A number of techniques including subtractive library screening, differential hybridization, representational difference analysis (RDA), mRNA differential display, serial analysis of gene expression (SAGE) and electronic subtraction have been developed for novel gene discovery to date. Subtractive library screening and differential hybridization are conventional methods [1]for the identification of molecules that differ in abundance between two pools of molecules. RDA [2, 3]and mRNA differential display [4]are polymerase chain reaction (PCR)-based techniques and are sufficiently sensitive for the detection of differentially expressed genes. SAGE is a technique that allows a rapid and detailed analysis of thousands of small expressed sequence tags (ESTs) [5, 6]. Electronic subtraction is the method comparing ESTs from experimental and control cDNA libraries [7–9], that also facilitates the identification of the abundant messages since the ESTs from a particular library only represent a small fraction of the total number of differentially expressed genes. Table 1 summarizes some basic features of these techniques for their utilization in novel gene discovery. In the last decade or so, these various techniques have been widely used to identify differentially expressed genes in many biological systems [10]. In particular, the mRNA differential display methodology has been adopted by a large number of laboratories as an important tool being applied for both in vitro and in vivo systems [11]. Detailed protocols for mRNA differential display have been described previously [12, 13]. In the present article, we briefly outline the critical procedure of the mRNA differential display technique and its technical improvements and modifications, and summarize its successful applications for the discovery of novel genes in normal and pathological functions of the cardiovascular system.


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  		Table 1 Comparison of the molecular techniques used for novel gene discovery

 

   2 Critical procedure of mRNA differential display and its technical improvements and modifications
Top
 Abstract
 1 Introduction
 2 Critical procedure of...
3 Applications of mRNA...
 4 Summary
 References
 
An overall strategic approach using mRNA differential display for novel gene discovery is outlined in Fig. 1. The critical technical procedure of mRNA differential display is described briefly below, relative merits, disadvantages, and the improvement and modifications of this technique are discussed along with the text. Table 2 summarizes some of the important modifications on mRNA differential display.


Figure 1
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  		Fig. 1 Schematic illustration of the experimental procedure for the discovery of disease-related genes using mRNA differential display.

 

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  		Table 2 Technical improvements/modifications of mRNA differential display

 
2.1 mRNA differential display
Messenger RNA (mRNA) is the product of gene expression that encodes for a specific protein. The levels of mRNA in the cell are generally reflected by transcriptional regulation. Following transcription, mRNA is ‘matured’ by capping the 5' end, polyadenylation [poly(A)] at the 3'-end, and splicing of the intron sequences in eukaryotic cells. Taking advantage of the polyadenylated tail present in most eukaryotic mRNA species, the mRNAs can be reverse-transcribed in the presence of anchored primers complementary to the 3'-end of mRNAs, such as the use of oligo-d(T)12–18 primers.

The method of mRNA differential display consists of two basic steps (Fig. 2): (1) reverse transcription using a set of 3'-anchored primers, such as T12MN [4]where M=G, A, C and N=G, A, T, or C, and (2) PCR amplification of cDNA fragments using arbitrary (upstream) primers and anchored (downstream) primers. For the RT reaction, total cellular RNA (usually DNase-treated to eliminate the possibility of genomic DNA contamination) is reverse-transcribed to yield the first strand cDNA primed with T12MN oligonucleotides. This RT reaction enables all the mRNA species having a poly(A) tail to be reverse-transcribed. Typically, this reverse transcription reaction is divided into 4 subgroups each using a different T12MN primer with G, A, T or C at the last base of the 3'-end. Because a large number of mRNA species are present in a cell, the division of subgroups for the RT allows a portion of the mRNA species to be displayed, which will increase the resolution of cDNA species after amplification [4].


Figure 2
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  		Fig. 2 Scheme of mRNA differential display. The classical technique is illustrated that consists of reverse transcription (A) and PCR (B). See the text for detailed descriptions on the technique and its modifications.

 
Amplification of all the cDNAs is carried out using an upstream arbitrary primer and a downstream anchored primer (identical to the one used for the RT) in the presence of a radioactive nucleotide (35S-{alpha}-dATP or 33P-{alpha}-dATP). The upstream primer has been optimized to 10 bases in length composed of approximately 50% of G or C base contents [4]. In addition, a relatively low annealing temperature (42°C) is applied in the PCR so that the number of the amplified mRNA species is maximized. Using these conditions of amplification, it has been estimated that at least 30–40 upstream primers in combination with the downstream primers will be necessary to amplify every mRNA species present in a given cell [25].

The amplified cDNA fragments are resolved by electrophoresis and subjected to autoradiographic analysis. By taking advantage of mRNA differential display, multiple samples can be amplified and compared in parallel. As such, differences in gene expression, either up- or down-regulated, can be identified in specific experimental or pathological conditions or along temporal expression patterns (Fig. 3A). For example, the differential display analysis was carried out using cellular RNAs isolated from LPS-stimulated and unstimulated rat carotid artery using a 5' arbitrary primer (5'-GACCGCTTGT-3') and a 3' T12MA or T12MG primer in the presence of [33P]dATP, and resolved by electrophoresis (Fig. 3B). Several bands were noted for their differential expression in response to LPS stimulation, and one band (indicated with an arrowhead in Fig. 3B) was identified for its differential regulation in the differential display gel and this observation was confirmed by Northern analysis [26].


Figure 3
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  		Fig. 3 Gel electrophoresis of differential display PCR products. A: Schematic illustration of the differential display PCR products after gel electrophoresis. The samples amplified from normal vs. disease specimen could be analyzed in parallel on a standard denaturing sequencing gel. The altered gene expression could be identified. For example, same genes are induced for their mRNA expression (indicated with arrowheads), while others may be down-regulated (arrow). B: Representative autoradiograph of differential display products for RNA isolated from cultured rat aortic artery stimulated with LPS. mRNA differential display was carried out using an upstream primer (5'-GACCGCTTGT-3') in combination with downstream primers (T12MA, M=G, A or C) in the presence of [33P]dATP, and the PCR products were resolved in an 8 M urea/6% polyacrylamide DNA sequencing gel in the following order: lane 1, unstimulated and lane 2, stimulated carotid artery from spontaneously hypertensive rats, lane 3, unstimulated and lane 4, stimulated carotid artery from Wistar-Kyoto rats. The band(s) indicated with an arrowhead shows a marked induction in response to LPS stimulation.

 
Since the initial description of mRNA differential display technique [4], a number of modifications have been made on the primers: (a) a single base anchored primer was described [14]that required less primers to generate similar amplification results; (b) a primer with an extended restriction enzyme site was used to facilitate subcloning and direct sequencing of the PCR products [27]; (c) longer primers (18–20 mers) for RT and PCR, in combination with two step PCR amplification (the first several cycles done at low annealing temperatures and the subsequent cycles at higher annealing temperatures that improved the fidelity of the amplification and reduced the incidence of false positive [15, 16, 22]). Additional modification has been made to overcome a strong bias towards high copy number mRNAs [23], using selected primers that preferentially isolate mRNAs of moderate to low abundance [24].

2.2 Band recovery and confirmation of the differentially expressed genes
Following mRNA differential display, the bands of interest may be recovered by applying the following steps: the DNA band is excised from the dried sequencing gel, isolated by extraction procedures, and reamplified using the same sets of primers as in the original PCR [4]. The recovered DNA band can serve as a probe to confirm mRNA expression by means of Northern blot analysis, and/or be subcloned into a vector for further analysis.

Confirmation of gene expression is one of the critical steps following mRNA differential display, in as much as a large number of false positive bands may be present on differential display. A variety of methods have been utilized in different laboratories to reduce false positives, of which the most commonly used method is Northern blot analysis. In addition, dot blot, quantitative RT/PCR, ribonuclease protection assays and other methods have also been used.

2.3 Sequence analysis of the differentially expressed genes
Sequencing of the differentially expressed cDNA is a logical next step. This step relies on DNA sequencing analysis of the recovered DNA band. Because multiple DNA species may be present in this recovered band and the differential display primers are too small to be used successfully for direct sequencing using standard protocols, the differential displayed DNA fragments are typically subcloned into a vector prior to sequencing analysis [4, 28]. Recent modifications using elongated primers for direct differential display [15, 16]or the reamplification following the original differential display method [27]allow direct sequencing analysis of the PCR products if only a single DNA species is present in a particular band.

Based upon the sequence information, the identity of the differentially expressed genes can be determined by searching a computer database, such as GenBank. If it represents a novel gene, a cDNA library can be screened using this DNA probe in order to obtain the full length cDNA clone.

It should also be noted that the cDNA fragments isolated by this method are in general small, and frequently located in the 3'-untranslated region (UTR). Therefore, in order to determine the identity of the differentially expressed gene, one may often require to isolate the full length cDNA clone

2.4 Functional analysis
The identification of the biological functions for the differentially expressed genes is one of the most challenging tasks. The gene may encode a secreted, cytoplasmic, or nuclear protein. To predict its function, one should first isolate the full length cDNA and predict the peptide sequence, and then explore the functional aspects based upon the encoding peptide. For example, the gene identified in Fig. 3B by differential display was further pursued for full length cloning and found to be a rat homologue of interferon inducible protein 10 (IP-10), a C–X–C chemokine [26]. Based upon structural and functional similarities of IP-10 to other cytokines and chemokines such as IL-1β and IL-8 (which have been demonstrated to involve SMC proliferation and migration) [29, 30]we hypothesized that IP-10 may have a role in vascular smooth muscle cell biology. IP-10 was then demonstrated to be a potent mitogen and chemotactic factor for vascular smooth muscle cells, two features of smooth muscle cell phenotypes that are believed to contribute to the pathogenesis of atherosclerosis and neointima formation [26].


   3 Applications of mRNA differential display in cardiovascular research
Top
 Abstract
 1 Introduction
 2 Critical procedure of...
 3 Applications of mRNA...
4 Summary
 References
 
Despite the relative advantages and disadvantages of mRNA differential display, it has been demonstrated as a powerful tool for novel gene discovery and investigation of the mechanisms involved in many biological and pathological functions. A number of laboratories have successfully applied this technique in cardiovascular research, including in vitro and in vivo studies which are summarized in Table 3.


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  		Table 3 Successful applications of mRNA differential display for novel gene discovery in cardiovascular function and diseases

 

   4 Summary
Top
 Abstract
 1 Introduction
 2 Critical procedure of...
 3 Applications of mRNA...
 4 Summary
References
 
mRNA differential display is one of the most flexible and comprehensive methods available for the detection of differentially expressed genes in cells and tissues. Since its initial description, this technique has been established in many laboratories and successfully applied for the identification of genes in in vitro and in vivo systems.

It also should be pointed out that similar to differential display, other methods, such as subtractive library screening, differential hybridization, electronic subtraction, SAGE and RDA, have also been successfully used for novel gene discovery. The application of these techniques will no doubt facilitate the discovery of novel therapeutic targets and help to understand the molecular mechanisms of disease. On the other hand, however, it should be pointed out that this is the first of many steps required in discovery of a novel therapeutic target especially as the function of the novel gene is likely to be unknown. Therefore, further actions should be taken to characterize the functions of the differentially expressed gene, including isolation of the full length cDNA, expression of the gene product for functional studies and target validation for the importance of this gene in disease processes.

Time for primary review 28 days.


   References
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 Abstract
 1 Introduction
 2 Critical procedure of...
 3 Applications of mRNA...
 4 Summary
 References
 

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