Introduction to Gene Expression Profiling
What is gene expression?
The central dogma of biology describes the method by which information is taken from genes and used to create proteins. DNA transcription produces RNA, then RNA translation makes proteins. This process is known as gene expression and all life forms use it to create the building blocks of life from genetic information .
A cell expresses only a selection of the genes it contains at any one time, which means that the cell can interpret its genetic code in different ways. Controlling which genes are expressed enables the cell to control its size, shape and functions. The ways in which an organism's cells express the genes they contain affects the organism’s phenotype, e.g. which color hair a mouse has, or whether it has hair at all .
What is gene expression profiling and who uses it?
Gene expression profiling measures which genes are being expressed in a cell at any given moment. This method can measure thousands of genes at a time; some experiments can measure the entire genome at once . Gene expression profiling measures mRNA levels, showing the pattern of genes expressed by a cell at the transcription level . This often means measuring relative mRNA amounts in two or more experimental conditions, then assessing which conditions resulted in specific genes being expressed.
Gene expression profiling is used by a variety of biomedical researchers, from molecular biologists to environmental toxicologists. This technology can provide accurate information on gene expression, towards countless experimental goals.
Different techniques are used to determine gene expression. These include DNA microarrays and sequencing technologies. The former measures the activity of specific genes of interest and the latter enables researchers to determine all active genes in a cell .
Once a genome has been sequenced, we know what potential a cell has—what characteristics and function it might have—based on the genes it contains. However, sequencing the genome does not tell us which genes a cell is expressing, or the functions or processes it is carrying out at any given moment. To determine these, we need to work out its gene expression profile. If a gene is being used to make mRNA, it is considered ‘on’; if it is not being used to make mRNA, it is considered ‘off’.
A gene expression profile tells us how a cell is functioning at a specific time. This is because cell gene expression is influenced by external and internal stimuli, including whether the cell is dividing, what factors are present in the cell's environment, the signals it is receiving from other cells, and even the time of day .
Why use gene expression profiling?
Gene expression profiling enables you to investigate the effects of different conditions on gene expression by altering the environment to which the cell is exposed, and determining which genes are expressed. Alternatively, if you already know a gene is involved in a certain cell behavior, gene expression profiling helps you to determine whether a cell is carrying out this function. For example, certain genes are known to be involved in cell division; if these genes are active in a cell, you can tell the cell is undergoing division, or whether a cell is differentiated [7,8].
Gene expression profiling is often used in hypothesis generation. If very little is known about when and why a gene will be expressed, expression profiling under different conditions can help design a hypothesis to test in future experiments. For example, if gene A is expressed only when the cell is exposed to other cells, this gene may be involved in intercellular communication. Further experiments could determine whether this is the case .
Gene profiling can also investigate the effect of drug-like molecules on cellular response. You could identify the gene markers of drug metabolism, or determine whether cells express genes known to be involved in response to toxic environments when exposed to the drug .
Gene profiling can also be used as a diagnostic tool. If cancerous cells express higher levels of certain genes, and these genes code for a protein receptor, this receptor may be involved in the cancer, and targeting it with a drug might treat the disease. Gene expression profiling might then be a key diagnostic tool for people with this cancer .
Different types of gene expression profiling
RNA expression patterns are key to predicting and classifying human disease based on specific biomarkers. To understand cellular responses, we must determine how gene expression changes are affected in relation to external stimuli, different environmental conditions and genetic lesions .
Transcriptome sequencing, using next-generation sequencing, lets us discover differentially expressed genes without requiring knowledge of which genes are involved .
Protein-coding RNAs are an important source of information, though non-coding RNA is also significant . Next-generation RNA sequencing enables such analysis, along with:
- ‘Digital counting’ of RNA molecules for highly quantitative and precise measurements
- Dynamic ranges to fully capture relevant biological changes
- The discovery of unknown RNAs (novel transcripts, splice variants, and gene fusions)
- The capture of all RNA types (poly-A+, long non-coding RNA and gene fusions) in a single assay
- Opportunities to focus, going from complete transcriptome analysis to a handful of pre-selected RNA sequences to balance experimental cost and ease of analysis with discovery potential
Quantification of mRNA using qPCR can be done using Applied Biosystems™ TaqMan® probe–based analysis and Applied Biosystems™ SYBR™ Green dye–based analysis plus using digital PCR, as discussed subsequently .
Although qPCR is useful for detecting gene expression changes of two-fold or more, a different approach is needed for measuring less than two-fold changes. Digital PCR (dPCR) can be used to resolve low-fold gene expression changes. dPCR enables:
- Absolute quantification of nucleic acid standards and next-generation sequencing libraries
- Rare target detection
- Enrichment and separation of mixtures
Knowing a cell is expressing certain genes provides a lot of information about how a cell is functioning, and potentially new insights into which genes (and therefore proteins) are involved in certain cellular behaviors. However, a gene does not code for just one protein . There are around 20,000 protein-coding genes in the human genome that produce many more proteins, probably in the order of 2 million . This is partly because cells use post-translational modification to change proteins after they have been created by the transcription-translation process, and because alternative splicing produces different proteins from the same gene .
We need more information than just the mRNA profile of a cell to establish a cell's function. It may be helpful, for example, to work out which proteins a cell makes through proteomics experiments . However, gene-expression profiling is still the best method to determine a cell's function from a single experiment.
When reporting gene expression profiling studies, it is typical to report the genes that had significantly different expression profiles under the experimental conditions. This is limiting because:
- Cellular differentiation means different cells express different genes at baseline; this is what gives them their different structures and functions.
- Many genes do not change because they are required in their original form for cell survival or are not affected by the experimental condition
- Altering levels of mRNA (measured during gene expression profiling) is not the only way in which a cell changes the genes it uses. Post-translational modification, for example, changes a protein that is made from the same gene. Changing mRNA levels are not necessarily associated with changing levels of protein .
Analysis of the data gathered in gene expression profiling experiments can be complex. However, expression profiling can give you information on the genes expressed under different conditions, enabling you to develop and test your hypothesis. Analyzing the data can be an interdisciplinary task and may require a biostatistician with multivariate statistical analysis to provide key support.
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For Research Use Only. Not for use in diagnostic procedures.