Gene Expression Profiling: What It Is and How to Do It

Biomedical researchers are turning to gene expression profiling to better understand the conditions under which certain genes are turned on or off — exploring the many variables that influence cellular function. Today, gene profiling studies are playing a valuable role in hypothesis generation and testing, in drug R&D as a method to pinpoint biomarkers, and even as a diagnostic tool in cancer treatment.

Let’s take a closer look at what gene expression profiling is and how it’s accomplished.

What is gene expression?

Inside a single cell, thousands of genes work to encode the proteins that determine how a particular cell operates. This information, stored inside our DNA, can be converted into instructions for making proteins or other molecules. In gene expression, DNA transcription is the process by which information in a DNA strand is copied into a new molecule of messenger RNA (mRNA). These mRNA copies of the gene are then used to trigger protein synthesis in ribosomes during the process of translation.

Gene expression is what we call the process of “switching on” a specific gene to start protein synthesis in ribosomes, which can then execute most of the active functions of a cell. Gene expression is controlled by a number of complex processes which determine if and when a gene is expressed. Gene expression, therefore, can be quite variable, depending on an assortment of biological and environmental conditions.

What is gene expression profiling?

Gene expression profiling is a laboratory method that measures mRNA levels at the transcription level – providing a snapshot of the genes in a cell or tissue being expressed in a given moment. A gene expression profile may include information about a cell's type, metabolic activity, and differential gene expression (which can be indicative of a disease state or cell response to an environmental stimuli.)

By altering the internal and external stimuli to which a cell is exposed, gene expression profiling allows biomedical researchers to investigate the impact of different influences on gene expression. As a result, gene expression profiling can be used to provide insights about specific cell behavior, and whether or not a particular gene is playing a role in that function. Gene expression profiles can also be used as a diagnostic tool to identify a disease, or as a method of determining a treatment’s efficacy.

How is gene expression profiling done?
There are several different methods commonly used in gene expression profiling. Among these techniques are DNA microarrays, which look at the activity of specific known genes; RT-qPCRs which are often used to validate microarray experiments; and RNA sequencing technologies, which allow researchers to determine all active genes in a cell at once.

  • DNA Microarrays – This technology is often used in gene expression studies to determine the extent to which certain genes are turned on or off in cells and tissues. In this method, a laboratory tool known as a microarray is used to detect the expression of thousands of genes at the same time, making it possible to retrieve an entire gene expression profile from a single experiment. To perform a DNA microarray analysis, reference and experimental samples are collected. The data gathered through these microarrays are then used to create gene expression profiles, which demonstrate any differentiation in response based on condition or treatment. Microarrays are most effective when being used to identify the expression of known genes and transcripts; they can’t be used for unidentified genes or transcripts.

  • RT-qPCR – Reverse-transcription quantitative PCR (RT-qPCR) is another key technology in gene expression analysis. RT-qPCR is both a routine and robust approach for measuring the expression of known genes or pathways of interest, validating microarray experiments in samples and monitoring biomarkers. Though qPCR is quite effective for low target numbers, its workflow can be less successful when sequencing high numbers of targets.

  • RNA Sequencing (RNA-Seq) Technologies – The high-resolution view of RNA-seq technologies allows for a more comprehensive understanding of gene expression. As a tool, these technologies — which can include mRNA sequencing, targeted RNA sequencing, and (sc) RNA-seq next-generation based (NGS) methods — are incredibly accurate and sensitive. These attributes give scientists previously unheard of visibility into disease states by offering full coverage, rapid profiling and deeper investigation of all active gene expression across the transcriptome (all of the mRNA molecules expressed from the genes of an organism). In contrast to microarrays, next-generation based RNA-Seq technologies can discover both known and novel features and reveal the entire transcriptome, versus only a few transcripts. RNA-seq technologies have especially broad application in drug discovery, where their high throughput, fast detection speed, simple analysis, and cost-efficiency are incredibly useful in drug screening and biomarker detection.

What is the importance of gene expression profiling?

Because gene expression profiling experiments allow researchers to measure the relative amount of mRNA expressed under differing conditions, they can be used to help demonstrate either a homeostatic response (the maintenance of equilibrium within a cell) or, alternatively, some kind of pathological condition.  

Increasingly, gene expression profile tests are being used to inform treatment decisions after diagnosis. For example, if breast cancer cells express higher levels of mRNA for a particular receptor than normal breast cancer cells, this could indicate to the research team that the receptor itself is playing a role in the breast cancer. Gene expression profiling can then be used to find a drug that interferes with this receptor, i.e. enabling identification or discovery of biomarkers for diagnosis, toxicity, or drug development. The potential benefits of these types of tests are significant: from more targeted treatment decisions to better patient outcomes.

In fact, gene expression profiling has had a huge impact on the investigation and management of breast cancer and other cancers. Recent efforts in this field have generated comprehensive molecular profiles of human cancers through groundbreaking RNA-seq technologies, fine tuning chemotherapy decision-making, improving delivery of treatments, and sparing many thousands of women from unnecessary toxic therapy. Today, genome profiling is considered a routine procedure in many cancer centers. Tumor genotyping is conducted and the resulting tumor genome profile is then used to determine therapy selection.

Understanding the why, when and how of gene expression is a complex science. Gene expression profiling gives biomedical researchers a critical tool for developing and testing their hypotheses, helping them form a deeper, more comprehensive picture of cell function and biology. Looking toward the future, emerging applications of RNA-sequencing tools are likely to continue to evolve gene expression profiling – driving the discovery of new disease biomarkers and therapeutic drug targets.

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