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Gene expression

Gene expression in the simplest terms can be defined as a process, by which inheritable information from a gene, such as the DNA sequence, is made or expressed into a functional gene product. However, the process is not so simple and straightforward. Several steps in gene expression may be modulated including the transcription step and the post-translational modification of a protein, thus, providing a strict regulation on expression levels of a particular gene.

With the availability of a huge amount of genetic information, science has entered the post-genomic era and one of the major challenges is revealing the function of genes and their products. This has led to an increased interest in Gene Expression Studies by researchers.

The collection of genes that are expressed or transcribed from genomic DNA, sometimes referred to as expression profile or transcriptome is a major determinant of cellular phenotype and function.

The transcription of genomic DNA to produce mRNA is the first step in the process of protein synthesis, and differences in gene expression are responsible for both morphological and phenotypical differences as well as indicative of cellular responses to environmental stimuli and perturbations.

Unlike the genome, the transcriptome is highly dynamic, and changes rapidly and dramatically in response to perturbations and even during normal cellular events such as DNA replication and cell division

The understanding of function of genes and knowing when and where and to what extent a gene is expressed is central to understanding the activity and biological roles of its encoded protein. In addition, changes in multigene patterns of expression can provide clues about regulatory mechanisms and broad cellular functions and biochemical pathways.

Hence, Gene Expression Studies has found its applications in diverse fields such as:

1. Gene discovery: In the identification of new genes, know about their functioning and expression levels under different conditions

2. Disease diagnosis: In understanding more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer.

Genetic disease is often caused by genes which are inappropriately transcribed—either too much or too little—or which are missing altogether. Such defects are especially common in cancers, which can occur when regulatory genes are deleted, inactivated, or become constitutively active.

Unlike some genetic diseases (eg. cystic fibrosis) in which a single defective gene is always responsible, cancers which appear clinically similar can be genetically heterogeneous.

For example, prostate cancer (prostatic adenocarcinoma) may be caused by several different, independent regulatory gene defects even in a single patient. In a group of prostate cancer patients, every one may have a different set of missing or damaged genes, with differing implications for prognosis and treatment of the disease.

Studying the expression levels of these heterogeneous genes can serve two purposes in studying cancer—it can pinpoint the transcription differences responsible for the change from normal to cancerous cells, and it can distinguish different patterns of abnormal transcription in heterogeneous cancers. Understanding the diverse basis of a cancer is crucial for inventing therapies targeted to the different varieties of the disease, so that each patient receives the most appropriate and effective treatment.

Though cancers are common examples of genetically heterogeneous diseases, they are by no means the only ones. Diabetes, heart disease, and multiple sclerosis are among the diseases for which genetic risk factors are known to be heterogeneous.

3. Drug discovery: Gene expression studies help determine the causes and consequences of disease, how drugs and drug candidates work in cells and organisms and what gene products might have therapeutic uses themselves or may have appropriate targets for therapeutic intervention.

Moreover, with the advancement in the field of gene expression studies, it will be possible for the researchers to classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.

4. Pharmacogenomics: This is a newly emerging field in the science of drug discovery. Scientists have already puzzled over as to why some drugs work better in some patients than in others? And why some drugs may even be highly toxic to certain patients? Pharmacogenomics is the hybridisation of functional genomics and molecular pharmacology. The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and the genetic profiles of patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesised by the diseased genes.

The researchers can use this information to synthesise drugs which combat with these proteins and reduce their effect.

5. Toxicological research: Toxicogenomics, a newly emerging science, establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. Gene expression studies provide a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny.

6. Cellular responses to the environment: Cells survive in the face of changes in temperature and pH, changing nutrient availability, and the presence of environmental toxins and ionising radiation. Usually, a change in environment requires that expression of some genes be turned up or down so that the organism can respond appropriately. Environmental changes of interest also include the introduction of signaling molecules, such as hormones, interleukins, and interferons, as well as the actions of drugs.

Relative gene expression studies can point out genes whose transcription changes in response to an environmental stimulus. Temporal studies can identify not only genes whose transcription changes but also the order of the changes, providing evidence about which genes control the response directly and which are only indirectly affected by it.

7. Cell cycle variations: Even in a stable environment, cells undergo DNA replication, mitosis, and eventually death. A cell's genes encode the "programs" for these activities, and gene transcription is required to execute those programs. Gene expression studies can be used to distinguish genes that are expressed at different times in the cell cycle. In this way, the pathways responsible for controlling basic life processes can be uncovered.

Hence, in view of above applications quantitative analysis of gene expression in from, sometimes a very limited amount of, RNA is of great interest to research scientists. Currently, researchers have been employing the techniques of Northern Blotting, Real Time PCR or MicroArray technology to determine mRNA levels for Expression studies.

For quantitative measurements, either Northern Blotting or Real Time PCR technologies are used. However, Northern Blotting requires the use of radioactive reagents and can have lower data quality than more modern methods (due to the fact that quantification is done by measuring band strength in an image of a gel), and of course a low throughput technique, but it is still often used. It does, for example, offer the benefit of allowing the discrimination of alternately spliced transcripts.

The other modern quantitative, though a considerably low-throughput approach for measuring mRNA abundance is Real Time Quantitative PCR. The Real Time PCR technology allows determination of quantitative gene expression levels wherein expression levels of more than one gene can be compared with a house keeping gene in a multiplex PCR in the same reaction tube. The lower level of noise in data obtained via qPCR often makes this the method of choice, but the price of the required equipment and reagents can be prohibitive.

Also, the present day instrumentations available in market makes the choice of multiplexing limited to a maximum of six color multiplexing only.

However, the DNA MicroArray technology provides an alternative in terms of high throughput gene expression studies. In this technology, transcript levels for thousands of genes at once (expression profiling) can be measured by spotting samples on a microarray slide or nylon substrates and subsequent hybridisation with fluorescent labeled probes. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously—a dramatic increase in throughput.

However, MicroArray technology is more a qualitative gene expression technology. It gives a lot of information of the expression levels of the various genes, but of not much help to a researcher trying to find answers in terms of quantitative gene expression studies.

Moreover, the microarray systems are expensive as they require special robotics for spotting DNA samples on the slides, and imaging equipments for scanning and reading the slides, and then sophisticated Data Mining software to process and analyse the data generated from the thousands of DAN samples.

Hence, a researcher who is interested to study the quantitative expression levels for a large number of genes, say 20 or more genes during biomarker validation, signal transduction or metabolic pathway studies has to first run his samples on a MicroArray platform, and then do a quantitative Real Time PCR running not more than six genes at one go. This becomes a tedious and expensive exercise for a researcher interested in doing quantitative gene expression studies for scores of genes.

Beckman Coulter has developed a new technology and has recently introduced the GenomeLab GeXP (Gene eXpression Profiler) Genetic Analysis System that can detect up to 30 genes in a single reaction containing 5 ng to 500 ng of total RNA. GeXP uses a patented universal priming strategy to overcome potential bias in amplified targets that are typically associated with other types of multiplexed assays.

GeXP offers the sensitivity, dynamic range and reproducibility of real-time methods while providing throughput gains, both in terms of numbers of genes per reaction and numbers of samples that can be efficiently processed. Unlike real-time methods that can monitor only a few genes at a time, XP is capable of simultaneously monitoring the expression levels of approximately 20 genes at a time. XP is able to achieve these higher levels of quantitation and multiplexing through the use of a proprietary priming strategy and single endpoint fluorescence detection.

The modified rtPCR process in GeXP technology uses a combined gene-specific, universal priming strategy that overcomes the primary deficiencies of rtPCR without compromising the detection sensitivity that is gained by using the process. The strategy is outlined in Figures 1 and 2.

Key to the process is the conversion of the multiplex amplification process from one involving tens of primers to one using only two primers. The reaction initialises using gene-specific primers that are capable of specifically detecting each target mRNA. These gene-specific primers carry on their 5' ends a consensus or universal sequence. During the first few cycles of amplification the specific gene targets are amplified by these chimeric primers, creating products that are tailed with the universal primer sequence.

The reactions all carry a pair of universal primers present at significantly higher concentrations. Therefore, as PCR progresses the amplification is quickly taken over by the single pair of universal primers. This transition from the use of many primers to only two effectively collapses the level of reaction complexity and locks in the relative concentrations of the different gene targets. In the universal primer amplification reaction all the products are effectively the same chemical species and are not differentially amplified.

There are some limitations in terms of the size range of PCR products, but the relative gene ratios can be maintained even as the reaction pushes into the plateau phase.

PCR products are analysed using the GeXP Capillary Electrophoresis System. Post amplification the different gene products can be differentiated and quantitated by capillary electrophoresis because each gene has been designed to generate a different size PCR product. Equipped with eight capillaries and a two-plate format, the GeXP system is capable of analysing 240 samples per 24 hour period. This produces 7,200 gene expression results for a 30-gene multiplexed assay. Researchers not only benefit from reduced labor and reagent expenditures, but also significant time-savings due to expedited data collection with the GeXP system.

Further, by providing more gene expression results per sample than real-time PCR, GeXP diminishes the experimental constraint imposed by limited quantities of RNA, such as those from formalin-fixed paraffin-embedded (FFPE) samples or tissue biopsies. The capabilities of GeXP have been increasingly recognised by academic and other research communities ranging from plant sciences and viral infection studies to cancer research.

The GeXP is really a versatile system, because in addition to its capability of doing quantitative multiplex gene expression studies, the system can perform De Novo sequencing, AFLP fingerprinting, SNP scoring, microsatellite instability, loss of heterozygosity, heterozygote detection, confirmatory sequencing and mutation analysis.

(Contributed by Beckman Coulter Product Management Team)