Gene expression is a highly regulated mechanism that controls the function and adaptability of all living cells including prokaryotes and eukaryotes. Several techniques exist for studying and quantifying gene expression and its regulation. Some of these techniques are old and well established while others are relatively new, multiplex techniques.
The field of gene expression analysis has undergone major advances in biomedical research. Traditional methods focused on measuring the expression of one gene at a time and not in any particular biological context. However, today, mRNA expression techniques have led to improvements in gene identification and disease sub-classification, for example.
Gene expression techniques
Analytical methods may be used to examine mRNA expression levels or differential mRNA expression. Some examples of these techniques are listed below.
Techniques that are low- to mid-plex
A gene contains two functional segments. One is a coding DNA sequence, which contains the instructions for making a protein. The other is a DNA sequence called a promoter, which is linked to this coding region and regulates the gene’s transcription, either by activating or suppressing its expression.
A reporter gene assay is used to determine the regulatory potential of a DNA sequence that is unknown. This involves a promoter sequence being linked to a detectable reporter gene such as luciferase, β-galactosidase or β-glucuronidase. Examples of methods used to determine the expressed reporter gene protein are fluorescence, absorbance and luminescence.
This is a technique used to detect specific RNA molecules present within an RNA mixture. Northern blotting is employed in the analysis of an RNA sample from a cell type or tissue so as to determine the RNA expression of certain genes.
Western blotting is a technique for detecting specific protein molecules within a protein mixture. This mixture might include all the proteins that are associated with a certain cell type or tissue. The technique can help to determine a protein’s size, and how much of it is expressed.
Fluorescent in situ hybridization (FISH)
This is a cytogenetic technique that can be used to identify and locate specific gene sequences. FISH can be used to visualize copy number aberrations such as the deletion, translocation or amplification of chromosomes. The technique is used in prenatal diagnosis and also provides a useful tool in the diagnosis and predicted prognosis of various sarcomas. The technique is also used in dermatology to help evaluate atypical moles.
Reverse transcription polymerase chain reaction (RT-PCR)
This is the most sensitive technique available for detecting and quantifying mRNA. Using RT-PCR, extremely small sample sizes can be used in the quantification of mRNA and the technique can in fact do this using just a single cell.
Techniques that are higher plex
Serial Analysis of Gene Expression (SAGE)
SAGE is a technique used to create a library of short sequence tags which can each be used to detect a transcript. The expression level of the transcript can be determined by assessing how many times each tag is detected. This technology enables comprehensive expression analysis across the genome.
Also known of as biochip or DNA chip, a DNA microarray is a solid surface to which a collection of microscopic DNA spots are attached. The microarrays are used to determine expression levels across a large number of genes or to perform genotyping across different regions of a genome.
This refers to methods used to measure the sequence of RNA molecules. Examples include shotgun sequencing of cDNA molecules acquired from RNA through reverse transcription and technologies used to sequence RNA molecules from a biological sample so that the primary sequence and abundance of each RNA molecule can be determined.
A tiling array is a type of microarray chip, with labelled DNA or RNA targets hybridized to probes attached to a solid surface. However, the probes used differ to those used with traditional microarrays. Rather than known sequences or predicted genes being probed, tiling arrays probe for sequences known to be present in a contigious region. This can provide information about regions that are sequenced but for which the local functions are largely unknown.