Multiplex (assay)
Encyclopedia
A multiplex assay is a type of laboratory
procedure that simultaneously measures multiple analyte
s (dozens or more) in a single assay
. It is distinguished from procedures that measure one or a few analytes at a time. Multiplex assays within a given application area or class of technology can be further stratified based on how many analytes can be measured per assay, where "multiplex" refers to those with the highest number of analyte measurements per assay (up to millions) and "low-plex" or "mid-plex" refers to procedures that process fewer (10s to 1000s), though there are no formal guidelines for calling a procedure multi-, mid-, or low-plex based on number of analytes measured. Single-analyte assays or low-to-mid-plex procedures typically predate the rise of their multiplex versions, which often require specialized technologies or miniaturization to achieve a higher degree of parallelization.
Multiplex assays are widely used in functional genomics
experiments that endeavor to detect or to assay the state of all biomolecule
s of a given class (e.g., mRNAs, protein
s) within a biological sample
, to determine the effect of an experimental treatment or the effect of a DNA mutation
over all of the biomolecules or pathway
s in the sample. The ability to perform such multiplex assay experiments measuring large numbers of biomolecular analytes has been facilitated by the completion of the human genome sequence
and that of many other model organism
s.
settings, where many specimens can be analyzed using a multiplex (or other) assay. Strictly speaking, a multiplex assay is not necessarily high-throughput. When the execution of a single multiplex assay generates data for a large number of analytes (e.g., gene expression
levels for all genes in the human genome
), it is considered high-throughput. However, it is more the ability to rapidly process multiple specimen
s in an automated fashion that characterizes high-throughput techniques. Massive parallelization of assays is one way to achieve "high-throughput" status. Another way is via automating
a manual laboratory procedure.
Laboratory
A laboratory is a facility that provides controlled conditions in which scientific research, experiments, and measurement may be performed. The title of laboratory is also used for certain other facilities where the processes or equipment used are similar to those in scientific laboratories...
procedure that simultaneously measures multiple analyte
Analyte
An analyte, or component , is a substance or chemical constituent that is of interest in an analytical procedure. Grammatically, it is important to note that experiments always seek to measure properties of analytes—and that analytes themselves can never be measured. For instance, one cannot...
s (dozens or more) in a single assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
. It is distinguished from procedures that measure one or a few analytes at a time. Multiplex assays within a given application area or class of technology can be further stratified based on how many analytes can be measured per assay, where "multiplex" refers to those with the highest number of analyte measurements per assay (up to millions) and "low-plex" or "mid-plex" refers to procedures that process fewer (10s to 1000s), though there are no formal guidelines for calling a procedure multi-, mid-, or low-plex based on number of analytes measured. Single-analyte assays or low-to-mid-plex procedures typically predate the rise of their multiplex versions, which often require specialized technologies or miniaturization to achieve a higher degree of parallelization.
Multiplex assays are widely used in functional genomics
Functional genomics
Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects to describe gene functions and interactions...
experiments that endeavor to detect or to assay the state of all biomolecule
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
s of a given class (e.g., mRNAs, protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s) within a biological sample
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...
, to determine the effect of an experimental treatment or the effect of a DNA mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
over all of the biomolecules or pathway
Metabolic pathway
In biochemistry, metabolic pathways are series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by a series of chemical reactions. Enzymes catalyze these reactions, and often require dietary minerals, vitamins, and other cofactors in order to function...
s in the sample. The ability to perform such multiplex assay experiments measuring large numbers of biomolecular analytes has been facilitated by the completion of the human genome sequence
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
and that of many other model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
s.
"Multiplex" versus "High-throughput"
Multiplex assays are often used in high-throughput screeningHigh-throughput screening
High-throughput screening is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening allows a...
settings, where many specimens can be analyzed using a multiplex (or other) assay. Strictly speaking, a multiplex assay is not necessarily high-throughput. When the execution of a single multiplex assay generates data for a large number of analytes (e.g., gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
levels for all genes in the human genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
), it is considered high-throughput. However, it is more the ability to rapidly process multiple specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...
s in an automated fashion that characterizes high-throughput techniques. Massive parallelization of assays is one way to achieve "high-throughput" status. Another way is via automating
Automation
Automation is the use of control systems and information technologies to reduce the need for human work in the production of goods and services. In the scope of industrialization, automation is a step beyond mechanization...
a manual laboratory procedure.
Nucleic acid-based multiplex techniques
- DNA microarrayDNA microarrayA DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
used for gene expressionGene expressionGene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
or SNPSingle nucleotide polymorphismA single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
detection assays - SAGESerial Analysis of Gene ExpressionSerial analysis of gene expression is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu...
for gene expression - High-throughput sequencing which can produce millions of short DNA sequences in parallel
- Multiplex PCR for applications requiring the amplification or sequencing of DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life.... - Multiplex Ligation-dependent Probe AmplificationMultiplex ligation-dependent probe amplificationMultiplex ligation-dependent probe amplification is a variation of the polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair. Each probe consists of a two oligonucleotides which recognise adjacent target sites on the DNA...
(MLPA) - DNA sequencing by ligationSequencing by ligationSequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase to identify the nucleotide present at a given position in a DNA sequence. Unlike most currently popular DNA sequencing methods, this method does not use a DNA polymerase to create a second strand...
Protein-based multiplex techniques
- Protein microarrayProtein microarrayA protein microarray, sometimes referred to as a protein binding microarray,provides a multiplex approach to identify protein–protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation, or to identify the targets of biologically active...
for measuring protein-protein interactions or small molecule binding - Antibody microarrayAntibody microarrayAn antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens...
a type of protein array in which antibodies are arrayed - Phage displayPhage displayPhage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...
for screening large protein libraries for interacting proteins or other molecules
Other multiplex techniques
- Tissue microarrayTissue microarrayTissue microarrays consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.-History:...
for analyzing multiple tissue samples - Cellular microarrayCellular microarrayA cellular microarray, is a laboratory tool that allows for the multiplex interrogation of living cells on the surface of a solid support. The support, sometimes called a "chip", is spotted with varying materials, such as antibodies, proteins, or lipids, which can interact with the cells, leading...
for observing cellular responses against a panel of materials - Chemical compound microarrayChemical Compound MicroarrayA chemical compound microarray is a collection of organic chemical compounds spotted on a solid surface, such as glass and plastic. This microarray format is very similar to DNA microarray, protein microarray and antibody microarray...
to assay multiple chemical compounds for specific activities - Multiplex detection enables the simultaneous detection of two or more targets on a western blot.
- Multiplex biomarker analysis of urine.
- ELISAELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
s are a type of assay that is not multiplex per se because each assay detects a single analyte, but it is typically parallelized via microtiter plateMicrotiter plateA Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
s to achieve high-throughput sample processing.