Gene expression refers to functional gene products (usually proteins) resulting from a specific gene being transcribed or “expressed”.
This is a multi-step process where gene regulation can occur at any step along the path including transcription, post-transcription, translation and post-translation.
Gene expression tells us how a cell is functioning at a given time. It is a process that allows a cell to respond to its environment. Knowing this is important because a specific protein can only be produced when its gene is turned on. Gene function involves the examination of when and where a gene is expressed in the cell or in the organism.
Regulating gene expression affects which proteins are expressed, when, and how much at any given time. This process is critical for cell differentiation and function. When these processes are altered, it can result in disease, making gene expression analysis is an important area of research.
Most gene expression is regulated at the transcriptional level. Regulatory proteins bind to DNA to affect transcription, so changes in expression of these proteins is one way that cells can regulate gene expression. Another mechanism for transcriptional regulation is controlling accessibility to the DNA binding site that initiates transcription. Repressor proteins, chromatin remodeling and epigenetic modifications all affect the ability of transcription factors to bind to the DNA and start transcription, thus affecting gene expression.
While most gene expression occurs at the transcriptional level, there are additional mechanisms that have been shown to be important in disease processes, such as post-transcriptional regulation. After transcription, RNA must then be transcribed into the functional protein to have an effect. Changing the integrity or stability of RNA can prevent protein translation and functional gene expression. miRNA are important post-transcriptional regulatory factors that result in silencing or degradation of the RNA altogether. In recent years, miRNA have been shown to play an important role in various disease processes including a variety of cancers.
Due to the involvement of gene expression in disease processes, several molecular methods have been developed. Since mRNA is an integral step in gene expression, researchers often measure mRNA levels to evaluate gene expression. One of the most common methods used is real-time PCR, which is also called real-time quantitative reverse-transcription PCR (qRT-PCR) or quantitative PCR (qPCR).
This procedure is based on PCR where DNA is amplified over multiple cycles allowing for detection of specific DNA sequences. Since the mRNA is being measured, reverse transcription must occur to generate complementary DNA which can then go through the PCR process.
Using fluorescence technology, small changes in gene expression can be detected and quantified. TaqMan and SYBR green are two of the most common markers used. TaqMan makes use of a gene-specific probe that contains a fluorescent dye that is quenched unless it binds to the specific sequence. SYBR green is a fluorescent dye that will bind to DNA. As the PCR process amplifies the gene-specific cDNA in a sample, the level of fluorescence increases with each cycle and is measured and recorded using a qPCR machine, like the Azure Cielo 6. The data can then be analyzed to determine gene expression in each sample.
Because qPCR can detect very small amounts of mRNA, this allows for gene expression analysis even on a very small sample amount, like with the COVID-19 detection assay. In fact, the Azure Cielo has superior detection sensitivity and is able to detect even one single copy of target viral DNA.
In addition to evaluating single gene expression patterns, multiple genes can be evaluated per sample as well using multiplex qPCR (shown in Figure 1). This method offers several advantages for investigators studying more than one target of interest. Multiplex qPCR assays can detect multiple targets in a single reaction, saving time and reducing sample requirements. More information is obtained per sample, which can especially be important when sample amounts are limited. Also, controls to account for pipetting differences between wells and plates aren’t needed because RNA levels or gene copy numbers are compared directly within the same reaction.
The Azure Cielo is a qPCR system designed to provide high-quality data through advanced and high-performance optical technology. Boasting broad-spectrum detection capability, exceptional specificity and precision, and faster run times, the Cielo consistently provides reproducible qPCR data. Learn more about the Cielo here.
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