Created by: Caty Chung, Nicolette Ross, Joan Glenny-Pescov, Jiaming Hu, Stephan Schurer*
Contact: Stephan Schurer stephan dot schurer at med dot miami dot edu
Commonly used statistical parameters for monitoring assay quality include Z and Z' factors. Prior to starting a large screen, and after assay optimization and miniaturization, pilot screens are performed to assess the quality of the assay run and to assess / validate the suitability of a assay for a high-throughput screening run.
Qualifies the endpoint as either the exact value (=) or less than (<) or greater than (>) the endpoint value specified. In case of less than or greater than, the endpoint cutoff is often determined manually; for example in a concentration response experiment the tested compound can be inactive at the minimum tested concentration; or a compound can still be active even at the lowest tested concentration.
cytokine-specific antibody bead-based method
This technology relates to measurement of protein and nucleic acid conformational properties. Proteins undergo changes to their tertiary structure upon heating, binding to perturbagens, other proteins and substrates. Nucleic acids have characteristic secondary structures, which include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine quadruplexes, triplexes, etc. Information about conformational properties of protein and nucleic acids can be obtained by circular dichroism spectroscopy, thermal shift, fluorescence resonance energy transfer (FRET), nuclear magnetic resonance (NMR), X-ray crystallography, etc.
Data normalization obtained by dividing the difference of mean of the positive controls and sample measurement by the difference of the mean of the positive controls and mean of the negative control.
In a viable cell, the phospholipid, phosphatidyl serine (PS) is localized on the inner leaflet of the cell membrane facing the cytosol. When the cell is undergoing apoptosis, the cell membrane loses its phospholipid asymmetry and exposes PS on the outer layer of the membrane. Annexin V specifically binds PS. Hence, apoptotic cells can be detected by staining with FITC conjugated Annexin V followed by flow cytometry.
Two kinds of inference errors can occur after screening at single perturbagen concentration: false positives and false negatives. To correct for this, the raw data is normalized using different statistical correction methods, namely, normalized percent distribution, Z-score, B-score, etc to derive at these endpoints.
Binding reporter technology is used to quantitate the interactions between two molecules, e.g. perturbagen-protein, protein-protein, protein-DNA, etc. The technologies used are radioligand binding, resonance energy transfer, fluorescence polarization, protein fragment complementation assay, and several label-free methods, including surface plasmon resonance, impedance, nuclear magnetic resonance, X-ray diffraction, isothermal titration calorimetry, mass spectrometry, etc.
It describes the different methods by which an enzyme activity can be measured and the ways it is expressed. This can be measured either directly or coupled to a secondary signal.
Inducible reporter technologies involve the use of a reporter gene to study the effect of perturbagens on gene expression. Some reporter genes can be easily detected, e.g. green fluorescence protein, and others function as selectable markers, e.g. antibiotic resistance.
molecular redistribution determination method
The technologies used to measure the distribution of organelles and molecules within the cell (proteins, second messengers, metal ions, dyes, etc). This is generally visualized by microscopy.
Compound cytotoxicity is an important parameter to measure when developing potential human therapeutics. Cytotoxicity is determined as a measure of radioisotope (3H thymidine or 51Cr) release, lactate dehydrogenase release from damaged cells, tetrazolium salt and alamar blue reduction, fluorescent dyes that selectively stain live or dead cells, and decrease in ATP content. The viability reporter technologies measure cell life or death by quantitating ATP content, caspase activity, membrane potential, and so on.
In situ refers to in place or position; undisturbed with respect to the antigen that is being tested in the immunoassay. The protein detection and localization is achieved by visualization under a microscope.
It measures the concentration of a substance in a solution based on the specific binding of an antibody to that substance, which produces a measurable signal. The signal measured depends on the label that is associated with this interaction, namely radioactive element (in radioimmunoassays), fluorescent, phosphorescent and chemiluminescent dyes, etc.
A competitive binding assay is based upon the competition of labeled and unlabeled ligand for a limited number of antibody binding sites. Briefly, the free antigen and antibody are incubated to form antigen-antibody complex and then the complex are bound to antigen-coated surface in the assay plate. The unbound antibody-antigen complex is washed off before adding enzyme-linked secondary antibody against the primary antibody. The substrate is then added and the antigen concentration can then be determined by the signal strength elicited by the enzyme-substrate reaction. In this assay, the enzyme-linked secondary antibody compete with the sample antigen which is associated with the primary antibody.
Enzyme linked immunosorbent assay or ELISA is used to detect the presence of a particular protein, either an antigen or antibody in the sample such as a body fluid.
An antigen-down immunoassay or immunometric assay involves binding the antigen to a solid surface instead of an antibody. Antigen-down immunoassays are used to bind antibodies found in a sample. When the sample is added (such as human serum), the antibodies (IgE for example) from the sample bind to the antigen coated on the plate.
direct enzyme activity measurement method
An enzyme is a protein that acts as a catalyst for specific biochemical reaction, converting specific substrates into chemically distinct products. Enzyme activity assays quantify the kinetics of the product formation or substrate depletion in the reaction, which result in fluorescence, luminescence or colorimetric outputs.
coupled enzyme activity measurement method
This technology is used to measure the effect of a perturbagen on an enzyme activity, which is coupled to a secondary signal. E.g., the activity of a kinase determines the residual pool of ATP, with ATP being its substrate, which in turn determines the rate of a coupled enzyme luciferase, since ATP is one of its substrates too.
Beta galactosidase gene is attached to the regulatory sequence of a gene of interest. The effect of a perturbagen on the expression of the gene of interest could be easily monitored by the detection of the product of beta galactosidase enzyme.
Beta lactamase gene is attached to the regulatory sequence of a gene of interest. The effect of a perturbagen on the expression of the gene of interest could be easily monitored by the detection of the product of beta lactamase enzyme.
Luciferase gene is attached to the regulatory sequence of a gene of interest. The effect of a perturbagen on the expression of the gene of interest could be easily measured by the detection of light emitted as a product the luciferase reaction.
Fluorescent proteins are commonly used as reporter genes to study the effect of perturbagens on gene expression. Examples include green fluorescent protein (GFP) from the jellyfish Aequorea victoria and the various modifications of it created by amino acid substitutions to either optimize the codon usage in mammalian cells or to change the spectral properties of the protein.
Cell viability can be assessed by the presence of cytoplasmic esterases that cleave a lipid-soluble nonfluorescent probe to yield a fluorescent product. The product is charged and is thus retained within the cell if the membrane is intact. Typical probes include fluorescein diacetate (FDA), carboxyfluorescein, and calcein.
Chromatin condensation is one of the morphological changes that occur during apoptosis. It is also known as pyknosis and can be visualized by staining with dyes followed by microscopy.
ATP levels are an indicator of viable cell number. ATP values higher than controls (untreated cells) indicate proliferation and cultures with ATP concentrations lower than controls indicate cytotoxicity. ATP levels can be measured using different commercial kits.
Caspases are cysteine proteases which play an important role in apoptosis. They are synthesized and stored in cells as inactive procaspases. Once apoptosis is initiated in a cell, the procaspases get activated by proteolytic cleavage. This sets in motion a caspase cascade, which leads to the activation of proteins involved in the chromatin fragmentation and finally culminates in cell death.
An expansion in the number of viable cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. The internal environment of the proliferating cell is more reduced than that of non-proliferating cells. Specifically, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD, increase during proliferation. Compounds such as tetrazolium salts and Alamar Blue, which can be reduced by these metabolic intermediates, can be used to monitor cell proliferation. Their reduction is accompanied by a measurable shift in wavelength with a corresponding change in color.
Live cells with intact membranes have the ability to exclude dyes that easily penetrate dead or damaged cells. Examples of the dyes include propidium iodide, 7-amino actinomycin D, and trypan blue. Cells stained with the former two can be quantified by flow cytometry, while the latter is by manual counting using hemocytometer.
Cellular nuclei can be by stained with Hoechst, DAPI, etc, which is a measure of cell viability and proliferation.
Live cells have a conserved and constitutive protease activity which serves as a biomarker of cell viability. The live-cell protease activity is measured using a fluorogenic, cell-permeant, peptide substrate (Gly-Phe-AFC). The substrate enters intact cells, where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells. The live-cell protease becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium.
This describes the type of alterations performed on the cell line, which include plasmid transfection, viral transduction, cell fusion, etc.
Cell fusion is the formation of a hybrid cell from two different cells of same or different species. After such fusion, one of the parental cells has a dominant phenotype over the other, and the resulting hybrids will have the features of only one of the parental cells. Fusion is performed to understand the dominance of genetic material, DNA recombination events, etc.
This refers to the introduction of cloned genes into cells by various methods, including transfection, transduction, electroporation, nucleofection, etc. It is generally performed to express the exogenously introduced gene and study its effect on the cell. In addition, DNA encoding regulatory moieties, such as siRNA is similarly introduced to inhibit the expression of a specific gene(s).
Cell fusion is the formation of a hybrid cell from two different cells of same or different species. Fusion cybrids are created by fusing a nucleated and an enucleated (nucleus removed) cell. The resulting cybrids have one nucleus, but they contain the mitochondria from both the parental cells. This allows one to study the mitochondrial inheritance and to ascertain the role of mitochondrial DNA mutations in diseases.
Cell fusion is the formation of a hybrid cell from two different cells of same or different species. Fusion hybrids are created by fusing two nucleated cells obtained from same or different species.
This refers to the infection of cells with either a pathogenic or a modified virus, which introduces viral genome into the cells. Pathogenic viruses include the influenza virus, measles virus, Japanese encephalitis virus, etc. For recombinant expression of proteins in cells or tissues, several types of viruses are utilized, including lentivirus, retrovirus, adenovirus, etc, with each having their own advantages and disadvantages.
This refers to the introduction of nucleic acids into cells using a construct. Plasmids constructs are created by the insertion of a gene or siRNA encoding DNA of interest into a plasmid. The engineered plasmids are introduced into cells by various means, namely, transfection, electroporation, nucleofection, etc which regulates the efficiency and location of gene transfer, for e.g., nucleofection delivers the plasmid directly into the nucleus in contrast to the other methods that deliver into the cytosol.
In this method, the introduced plasmid construct is stably integrated in the cellular genome, and is hence a permanent modification of the cells. This is achieved by selecting for cells which express a plasmid-encoded selectable marker, which is commonly an antibiotic resistance protein.
It is the temporary introduction of a gene or siRNA encoding DNA of interest into a cell using various plasmids. Usually, the transiently transfected DNA remains in the cell for 2-3 days and is functional: expresses the gene of interest. There are numerous transient transfection reagents and protocols available that have been optimized for various cell lines.
This describes the method by which a protein is purified from its source.
This involves cloning the cDNA of the gene of interest in a plasmid or viral vector and introducing it into a bacterial, insect or mammalian cell for expression. The expressed protein is then purified after lysing the cells followed by a series of centrifugation and, or chromatography and electrophoresis methods.
All living cells maintain a resting membrane potential, which is the difference in voltage between the interior and exterior of the cell. Electrically active cells, such as neurons, muscle and endocrine cells undergo changes in membrane potential due to depolarization and hyperpolarization as a result of opening and closing of ion channels. Action potentials are large and rapid changes in membrane potential which are generated as a result of a large depolarization event. In neurons, action potentials are used to transmit signals across neurons, while in muscle and endocrine cells, they lead to intracellular events, such as muscle contraction and hormone release, respectively. In addition, mitochondria possess a trans-membrane potential as a result of their respiratory function. Membrane potential can be measured either by using electrophysiological techniques or by staining with dyes whose binding is dependent on the membrane potential.
It is used for analysis of receptor-ligand interactions in screening applications. It utilizes a radiolabeled ligand and a source of receptor (membranes, soluble/purified).
This is used to study the interaction of two proteins. An enzyme or fluorescent protein is rationally dissected into two fragments and fused to two test proteins, whose interaction is being studied. Binding of the two test proteins results in the reconstitution of the enzyme or fluorescent protein from the two fragments, which can be measured.
This technique is used to detect proteins and peptides in a tissue section by binding to specific antibodies. Subsequently, a secondary antibody that is conjugated to a fluorophore or an enzyme needed for the detection of a signal is hybridized to the primary antibody. Signal is generally detected by immunofluorescence or less commonly, by a colored reaction. This allows the detection and subcellular localization of the protein of interest by visualization under a microscope.
This technique is used to detect proteins and peptides in a cell by binding to specific antibodies. Subsequently, a secondary antibody that is conjugated to a fluorophore or an enzyme needed for the detection of a signal is hybridized to the primary antibody. Signal is generally detected by immunofluorescence or less commonly, by a colored reaction. This allows the detection and subcellular localization of the protein of interest by visualization under a microscope.
This refers to the correction performed on the raw data values from an assay. B-score correction is a common method used for this purpose.
mitochondrial membrane potential assessment
Activity of the mitochondrial respiratory chain results in the generation of a proton gradient which consists of two components: a membrane potential and a pH gradient. Disruption of delta-psi has been shown to be one of the first intracellular changes following the onset of apoptosis. Delta-psi is generally quantitated by staining with specific dyes whose binding is dependent on the potential and less commonly, by electrophysiological techniques.
Electrically active cells, such as neurons, muscle and endocrine cells undergo changes in membrane potential due to depolarization and hyperpolarization as a result of opening and closing of ion channels. The electrical recordings from these cells are obtained by the insertion or attaching microelectrodes to these cells. Patch clamp, voltage clamp and current clamp are three ways of studying the current-voltage relationships of these ion channels.
Most often, the mitochondrial membrane potential which is called delta-psi is quantitated by staining with specific dyes whose binding is dependent on the potential.
The (raw) measurement is normalized as a percentage of the positive control. This is typically done by plate.
Curve fit methods include Levenberg-Marquardt, Simplex, Linear Regression (linear least-squares), etc. The Levenberg-Marquardt method is a robust algorithm and has become the standard for non-linear regression.
The technologies used to measure the size and shape of cells and organelles (phenotype). These are detected by microscopy.
Following a western analysis, the individual proteins in a gel, blot, or autoradiogram are quantitated by a densitometric scan over a limited range of protein concentration per band (1-10 ug). A scanner measures the optical density of the material by directing a beam of light and measuring its transmission or reflection.
Ex situ refers to out of place or position, with respect to the antigen that is being tested in the immunoassay. Usually the antigen is present in a tissue or cell lysate, or as a purified protein in the assay.
A digital system used for quantitating proteins in gels, blots, etc.
This technology relates to measurement of nucleic acid conformational properties using one of the following methods: circular dichroism spectroscopy, thermal shift, fluorescence resonance energy transfer (FRET), nuclear magnetic resonance (NMR), X-ray crystallography, etc.
This technology relates to measurement of protein conformational properties using one of the following methods: circular dichroism spectroscopy, thermal shift, fluorescence resonance energy transfer (FRET), nuclear magnetic resonance (NMR), X-ray crystallography, etc.
chloramphenicol acetyltransferase induction
Chloramphenicol acetyltransferase (CAT) gene is attached to the regulatory sequence of a gene of interest. When induced to express, CAT breaks down the antibiotic chloramphenicol resulting in a resistance against that antibiotic. The effect of a perturbagen on the expression of the gene of interest could easily be monitored by the gain of antibiotic resistance by the cells.
The technologies used to measure the movement of cells and organelles (phenotype). These are detected by microscopy.
It describes the assay dynamic range considering range and data variation: Z=1-{3x[StdDev(sample)+StdDev(negcontrol)]/[Avg(sample)-Avg(negcontrol)]} Not to be confused with z-score.
It describes the assay dynamic range considering range and data variation: Z'=1-{3x[StdDev(poscontrol)+StdDev(negcontrol)]/[Avg(poscontrol)-Avg(negcontrol)]}
Difference of signal between positive and negative controls; assay dynamic range: SW={Avg(poscontrol)-Avg(negcontrol)-3x[StdDev(poscontrol)+StdDev(negcontrol)]}/StdDev(poscontrol).
Measure of signal strength. S/N=[Avg(poscontrol)-Avg(negcontrol)]/[StdDev(poscontrol)+StdDev(negcontrol)]
Measure of the signal dispersion: CV=100x[StdDev(sample)/Avg(sample)]
This refers to the infection of cells with a modified virus, which introduces viral genome containing the foreign genes. Several types of viruses are utilized for recombinant gene expression, including lentivirus, retrovirus, adenovirus, etc, with each having their own advantages and disadvantages.
This refers to the infection of cells with a disease-causing virus, which could effect humans, animals and plants. Pathogenic viruses include the influenza virus, measles virus, Japanese encephalitis virus, etc.
It is a live cell assay in which eukaryotic cells are infected with bacteria.
Cells are counted to determine their viability. Cultured animal cells are counted either manually using hemocytometer, or by an automated cell counter, e.g. Coulter counter or Vi-CELL. The latter can distinguish live and dead cells by the trypan blue cell exclusion method. Yeast and bacterial cell growth is determined by measuring absorbance in a spectrophotometer, or by viable plate count.
This describes whether the perturbagens are tested individually or as mixtures / pools
The perturbagens are tested individually in an assay
Mixtures / pools of perturbagens are tested in an assay
cell cycle progression assessment method
In molecular biology, the polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR) or kinetic polymerase chain reaction, is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.
Reverse transcription polymerase chain reaction (RT-PCR) is a variant of PCR in which the RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional or real-time PCR.
Quantitative reverse transcription PCR is a PCR technique used to determine the amount of cDNA in a sample. It is the most commonly used form of quantitative PCR (qPCR). This technique is also called real-time reverse transcription PCR.
The direction of the readout signal, relative to negative controls, which is considered to be of interest for determining active perturbagents (hits).
A parameter defining the separation of the noise bands of the negative and positive controls in an experiment using robust statistics. The robust z' is defined as 1-(3*(MAD(pos)-MAD(neg))/abs(median(pos)-median(neg)) where MAD(pos) is the median absolute deviation of the positive control population and MAD(neg) is the median absolute deviation of the negative control population. Typically defined on a per-plate basis but may be defined for an overall assay.
RNA interference construct preparation method
The assay design method describes how a biological or physical process screened / investigated in the model system is translated into a detectable signal. This relates to the technology / technologies used to make the assay system work, i.e. enable that the screened process can be detected. It typically involves some manipulation of the (biological) model system to detect the process of interest.
Enzyme multiplied immunoassay technique, or EMIT, is a common method for screening urine and blood for drugs, both legal or illicit. A mix and read protocol has been developed that is exceptionally simple and rapid. The most widely used applications for EMIT are for therapeutic drug monitoring (serum) and as a primary screen for abused drugs and their metabolites (urine).
structure activity relationship analysis
In a conventional 2-site enzyme immunoassay, the antigen under assay which must have two or more epitopes, is insolubilised by reaction with an unlabelled antibody conjugated to a solid phase and reacted with an enzyme-labelled antibody directed to a different (preferably roomly-spaced) epitope of the analyte. The quantity of labelled antibody which becomes immobilised due to the complexing reaction is directly proportional to the amount of analyte present in the sample.
Titration is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of a known reactant.
Karl Fischer titration is a classic titration method in analytical chemistry that uses coulometric or volumetric titration to determine trace amounts of water in a sample. It was invented in 1935 by the German chemist Karl Fischer.
Dot immunobinding assay (Dot Iba) is a simple and highly reproducible immunodiagnostic method. Antibody or antigen is dotted directly onto nitrocellulose membrane (NCM) discs. The diagnostic material to be checked can be incubated on this disc. Presence of antigen antibody complex in NCM discs can be directly demonstrated with enzyme-conjugated antiglobulins and substrate. Development of a purple-pink colored, insoluble substrate product in the NCM will be considered a positive result in the assay. This assay allows the processing of multiple specimens at a time.
A checkerboard titration is a single experiment in which the concentration of the two components is varied in a way that will result in a pattern. This method is often used to optimize reagent concentrations and to measure susceptibility to microbes to various agents.
chemiluminescence-linked immunosorbent assay
The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. ELISPOT assays were originally developed to enumerate B cells secreting antigen-specific antibodies, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.
Fluorescence-linked immunosorbent assay (FLISA) is a technique where a fluorescent probe is used on an antibody of interest, which results in direct detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient.
The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to antibody, since at least two antibodies act in the sandwich. So sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein.
The In-Cell Western Assay is an immunocytochemical assay that uses NIR fluorescence to detect and quantify proteins in fixed cells. Detecting proteins in their cellular context increases quantification precision. Proteins in fixed, cultured cells are detected directly in microplates, which yields higher throughput compared to Western blotting and eliminates typical Western blotting steps such as cell lysate preparation, electrophoresis, and membrane transfer. Using the In-Cell Western Assay kits, the cost per well for secondary screening is reduced to a fraction of the cost of typical western blots.
The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
Immunolabeling is a means of localizing particular antigens within tissue. Immunolabeling is a methodological process where: 1) antibodies are used to identify antigens within an organism and 2) a tag (e.g. through fluorescence, gold beads, epitope tag, etc.) is added to a secondary antibody which binds to the primary antibody.
Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes.
Immunolabeling method in which gold particles are conjugated to a primary or secondary antibody and used for identification of an antigen.
Assay supporting methods describe the methods to prepare, generate or post-process the assay system or assay screening process. Such methods include the sample preparation, labeling, data processing, etc., which are required to performing the assay, but do not constitute the actual assay (design method).
Serial analysis of gene expression (SAGE) 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.
A Knock-in refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome. The difference between knock-in technology and transgenic technology is that a knock-in involves a gene inserted into a specific locus, and is a "targeted" insertion.
Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification (a change in the DNA of one of the organism's chromosomes) or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene.
Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene.
gene knockdown by lentiviral shRNA transduction
Antisense RNA is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery.
Method for reducing gene expression using morpholinos. Morpholino oligomers are an antisense technology used to block access of other molecules to specific sequences within nucleic acid. Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid (RNA).
A gene knockout is a genetic technique in which an organism is engineered to carry genes that have been made inoperative (have been "knocked out" of the organism).
Gene overexpression refers to genetic techniques in which an organism or cell line is engineered to express increased levels of a gene.
gene overexpression by stable transfection
gene overexpression by transient transfection
single nucleotide polymorphism genotyping
A method that uses purified enzymes to isolate and then replicate specific nucleic acids to levels where they can be detected. Examples are PCR, TMA, NASBA, etc.
Mapping is the construction of a series of chromosome descriptions that depict the position and spacing of unique, identifiable biochemical landmarks, including some genes, that occur on the DNA of chromosomes.
Method in which the amount of a substance is determined by measuring the volume of a second substance that combines or reacts with the first in known proportions.
The the MTS assay is a laboratory test and a standard colorimetric assay (an assay which measures changes in color) for measuring the activity of enzymes that reduce MTS + PMS to formazan, giving a purple color. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a water-soluble formazan product that has an absorbance maximum at 490-500 nm in phosphate-buffered saline. It is advantageous over MTT in that (1) the reagents MTS + PMS are reduced more efficiently than MTT, and (2) the product is water soluble, decreasing toxicity to cells seen with an insoluble product.
The MTT assay is a colorimetric assay that relies on the enzymatic reduction of a yellow tetrazolium salt, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), which forms a purple formazan crystal in metabolically active cells. The formazan can then be solubilized producing a concentration dependent colorimetric signal at 570 nm proportional to the cell number and activity.
The XTT Cell Proliferation Assay is a colorimetric assay system which measures the reduction of a tetrazolium component (XTT) into soluble formazan product by the mitochondria of viable cells. The samples are read using an ELISA plate reader at a wavelength of 450nm. The amount of color produced is directly proportional to the number of viable cells.
For spectrophotometric measurement of metabolic activity of living cells. Absorbance of converted dye is measured at a wavelength of 600 nm. Bioreduction of the dye reduces the amount of its oxidized form (blue) and concomitantly increases the fluorescent intermediate (red).
The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content.
The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when the previously red form coomassie reagent changes and stabilizes into coomassie blue by the binding of protein.
Coomassie dyes (also known as Coomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels.
The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques.
Ponceau S, Acid Red 112, or C.I. 27195 is a sodium salt of a diazo dye that may be used to prepare a stain for rapid reversible detection of protein bands on nitrocellulose or PVDF membranes (Western blotting), as well as on cellulose acetate membranes. A Ponceau S stain is easily reversed with water washes, facilitating subsequent immunological detection.
The bicinchoninic acid assay (also known as the BCA assay or Smith assay) is a biochemical assay for determining the total level of protein in a solution, similar to Lowry protein assay, Bradford protein assay or biuret reagent. The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using colorimetric techniques.
Crystal violet is a dye derived from gentian violet that is used as a general biological stain and an acid-base indicator.
fluorescent DNA hybridization probe method
A technique for DNA detection using SYBR Green 1 stain.The fluorescence intensity of SYBR Green is enhanced over 100-fold on binding to DNA which results in bright fluorescent DNA bands against a very dark background. Using a Dark Reader transilluminator, it is possible to detect less than 100 pg of SYBR Green-stained DNA by eye and tens of picograms using a CCD system.
Ethidium bromide is an intercalating agent used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for various techniques. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA.
The assay utilizes the displacement of ethidium bromide (or thiazole orange) from hairpin deoxyoligonucleotides.
Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain DNA. When excited by 488 nm of laser light, it can be detected with 562-588 nm band pass filter.
A technique of attaching a label to the active site of a large molecule and especially a protein by using a molecule which binds loosely and reversibly to the large molecule's active site and which has an inactive part that can be converted by photolysis to a highly reactive form causing the complex of the large substrate molecule and the loosely attached label to bind more permanently.
Method that employs a stain that is capable of entering and staining a living cell without causing an injury.
Dye staining method in which the dye is excluded from particular cells, usually due to the presence of an intact plasma membrane.
Dye uptake assays using neutral red, trypan blue and fluorescent compounds such as propidium iodide are traditional methods to assess cell viability and membrane damage. Trypan blue and fluorescent compounds such as propidium iodide are excluded by healthy, functional cell membranes but are able to traverse damaged cell membranes thereby preferentially staining dead cells.
Refers to variations on the two-hybrid technique, which measures activation of a reporter gene caused by binding between fusion proteins or other molecular entities.
The one-hybrid variation of the hybrid screening technique is designed to investigate protein-DNA interactions and uses a single fusion protein in which the AD is linked directly to the binding domain.
RNA-protein interactions have been investigated through a three-hybrid variation of the two-hybrid technique. In this case, a hybrid RNA molecule serves to adjoin together the two protein fusion domains which are not intended to interact with each other but rather the intermediary RNA molecule (through their RNA-binding domains).
A molecular biology technique used to discover protein-protein interactions and protein-DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively. The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For two-hybrid screening, the transcription factor is split into two separate fragments, called the binding domain (BD) and activating domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for the activation of transcription.
Two-hybrid assay performed in mammalian cells.
Parallel artificial membrane permeability assay
Parallel artificial membrane permeability
The process of constructing non-native membrane like structures from synthetic or natural amphiphilic molecules, polymers, surfactants, or modified macromolecules that mimic the composition, architecture, and functional properties of biological membranes.
using capture antibodies which are able to isolate large enzyme complexes in the their fully intact, fully active states from small samples of tissue or cells.
Test based on a strip of paper coated with an immobilized antibody specific for an antigen that is characteristic of a disease. Samples conjugated to a second antibody (which allows chromogenic detection) can be applied to the paper strip and results visualized in less than 10 minutes. By using multiple antigens at different positions on the strip, multiple diseases can be screened for in one sample.
The removal of certain proteins by the use of antibodies.
It is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.
chaperone coupled enzyme activity measurement method
The effect of a perturbagen on an enzyme activity is coupled to a secondary signal. The activity of an enzyme, e.g. luciferase is coupled to the effect of perturbagen upon the function of a chaperone, e.g. Hsp90, as denatured luciferase could serve as a substrate for Hsp90 mediated refolding.
protease coupled enzyme activity measurement method
The effect of a perturbagen on an enzyme activity is coupled to a secondary signal. The activity of an enzyme, e.g. phospholipase A2 is dependent on the effect of perturbagen upon the function a protease, e.g. ubiquitin-specific protease-2.
substrate coupled enzyme activity measurement method
The effect of a perturbagen on an enzyme activity is coupled to a secondary signal. The activity of an enzyme, e.g. luciferase is limited by the availability of one of its substrates (ATP or luciferin), which is coupled to the activity of another enzyme, e.g. a kinase or Cytochrome P450.
ATP coupled enzyme activity measurement method
The effect of a perturbagen on an enzyme activity is coupled to a secondary signal. The activity of an enzyme, e.g. luciferase is limited by the availability of one of its substrates, namely, ATP, which is coupled to the activity of another enzyme, e.g. a kinase, ATPase, etc.
luciferin coupled enzyme activity measurement method
The effect of a perturbagen on an enzyme activity is coupled to a secondary signal. The activity of an enzyme, e.g. luciferase is limited by the availability of one of its substrates, namely, luciferin, which is coupled to the activity of another enzyme, e.g. a Cytochrome P450, protease, etc.
protein G-based affinity purification method
antigen-based affinity purification method
protein A-based affinity purification method
protein A/G-based affinity purification method
protein L-based affinity purification method
cofactor coupled enzyme activity measurement method
luciferase enzyme activity measurement method
Raw sample measurements are normalized relative to positive and negative control samples with known activity and inactivity. Endpoints are usually expressed as percentage, which include percent activity, percent inhibition, etc.
Where control samples are not available, data can be normalized statistically assuming that the majority of the samples are negative, i.e. not active. An example is z-score.
Methods to process the measurement into the result type / endpoint reported as the final outcome of the assay; or other characteristics of the assay, which as data characterizing quality of an assay. Includes endpoint normalization and assay quality assessment.
ADP coupled enzyme activity measurement method
second messenger competitive binding assessment method
The technologies used to quantify the expression level of the entire gene pool in a cell or a subpopulation (e.g. tissue-specific gene expression, kinases pool, etc.) using one of the three methods, namely: microarray, bead based method or RNASeq.
phosphoprotein-specific antibody-coated bead based method
Gene-specific DNA is immobilized by spotting on a solid support, such as a glass slide. The DNA is hybridized with labeled nucleic acids from a sample and the signals measured, which reflect the composition of the sample nucleic acids. This is performed to quantify gene expression under various situations,e.g. diseases, development, etc.
RNA-Seq is an approach for transcriptome profiling which uses deep-sequencing technologies. RNA-Seq, also called "Whole Transcriptome Shotgun Sequencing" ("WTSS") and dubbed "a revolutionary tool for transcriptomics", refers to the use of High-throughput sequencing technologies to sequence cDNA in order to get information about a sample's RNA content, a technique that is quickly becoming invaluable in the study of diseases like cancer. Thanks to the deep coverage and base level resolution provided by next-generation sequencing instruments, RNA-Seq provides researchers with efficient ways to measure transcriptome data experimentally, allowing them to get information such as how different alleles of a gene are expressed, detect post-transcriptional mutations or identifying gene fusions.
The methods required to perform typical screening assay. These include the assay design methods (to generate a detectable signal) and assay supporting methods. Assay methods include laboratory methods. Computational methods are not currently categorized as assay methods, although some may also be used to process the assay measurement results.
nucleic acid identification and quantitation method
flat field correction; shading correction
Background correction is a data transformation designed to remove irrelevant contributions from the measured signal, e.g. those due to instrument noise or sample preparation.
Image resize is a process to change the size of the image data by increasing or decreasing the number of pixels within the image. Mostly used for reducing in either the horizontal or vertical dimension.
Image sharpening is a process to enhance the definition of object edges in an image.
Image smoothing is a set of local pre-processing methods designed to suppress image noise.
Edge detection is the process to identify regions in a digital image where the image brightness changes sharply or the image has discontinuities.
Image thresholding is a procedure that eliminates shades of gray from regions of an image above or below particular gray levels, replacing them instead with solid white or black.
Any method that simply measures the overall abundance (concentration, relative amount, etc) of a particular molecule (lipids, second messenger, nucleic acid, protein, etc.)
Second messengers relay and amplify signals from receptors to the cell interior as part of a signaling event. Examples include cAMP, cGMP, calcium, nitric oxide, diacylglycerol, etc. Levels of these second messengers are measured to reflect strength of the signaling.
Measuring lipid abundance could include mass spec-based methods and lipid droplet formation, a fluorescent intensity+image analysis assay to measure lipid stored in droplets within a cell,
Cell density, typically measured by absorbance (often indicated as A600 or OD600), is often used for bacterial or fungal cultures as an indication of cell number, viability or proliferation.
Beta-galactosidase activity measurement method
Any method that simply measures the overall abundance (concentration, relative amount, etc) of a nucleic acid (DNA or RNA), typically via hybridization methods
Any method that simply measures the overall abundance (concentration, relative amount, etc) of a small molecule, such as LC-MS/MS or LC-UV. Typically used in both in vitro and in vivo ADME assays to monitor compound.
Any method that measures the size and shape of whole cells. Typically detected by microscopy or flow cytometry
sub-cellular morphology assessment method
Any method that measures the size and shape of structures or organelles within cells, such as nuclei, mitochindria, Golgi, ER or vesicles. Typically detected by microscopy or flow cytometry
Any study performed in a whole, living organism (usually an animal such as a mouse or rat).
Any study performed in a whole, living organism (usually an animal such as a mouse or rat), to test the effects of a treatment on a specific disease modeled in this animal. Study design and measured endpoints vary tremendously and depend on the particular aspects of the targeted disease. Ideally, the endpoints measured will correlate with how efficacy would be measured in humans.
Any study performed in a whole, living organism (usually an animal such as a mouse or rat) to investigate the pharmacokinetics (PK), pharmacodynamics (PD) or toxicity of a perturbagen or treatment.
Changes in protein thermal stability can be induced by ligand binding, and this thermodynamic stabilization can be measured by a variety of methods, often reported as a delta Tm (or change in the melting temperature of the protein).
A method that uses chemilumiscence to quantify the labeled entity. For this, an enzyme converts a substrate to a product that emits photons of light
A method that is used to identify the precise order/sequence of the nucleotides in a dna molecule by using the shotgun method in which DNA is fragmented and then the individual frgaments are sequenced
A method that is used to identify open / accessible genomic sites of remodeled chromatin
A method that is used to quantify various cell metabolites or metabolism-related analytes (e.g. O2, glucose, pH)
epigenetic modification detection method
A method that is used to identify the DNA regions that are being occupied by a predefined transcriptional factor(s)
Techniques used to separate mixtures of macromolecules based on physical size and mass
NanoBRET (Nano Bioluminescence Resonance Energy Transfer) is a proximity-based assay that uses the bright, small luciferase NanoLuc (Nluc), fluorescent molecule (NCT ligand) and its substrate furimazine to monitor molecular interactions, protein conformational changes, or ligand binding in live cells.
the lysine ligand covalent binding method is used to measure binding of an amine-reactive molecule with a lysine residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
methionine ligand covalent binding method
the methionine ligand covalent binding method is used to measure binding of an thioether-reactive molecule with a methionine residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
the serine ligand covalent binding method is used to measure binding of an alcohol-reactive molecule with a serine residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
the tyrosine ligand covalent binding method is used to measure binding of a phenol-reactive molecule with a tyrosine residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
N-terminal ligand covalent binding method
the N-terminal ligand covalent binding method is used to measure binding of an amine-reactive molecule with the N-terminus of a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
threonine ligand covalent binding method
the threonine ligand covalent binding method is used to measure binding of an alcohol-reactive molecule with a threonine residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
unnatural amino acid ligand covalent binding method
the unnatural amino acid ligand covalent binding method is used to measure binding of a reactive molecule with a particular unnatural residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology. Unnatural amino acids can contain a variety of ‘bioorthogonal’ chemical handles.
the cysteine ligand covalent binding method is used to measure binding of a thiol-reactive molecule with a cysteine residue on a protein of interest. Binding assessment can be measured by protein mass spectrometry or other direct-binding methodology.
liquid chromatography-tandem mass spectrometry
A method wherein liquid chromatography is coupled to tandem mass spectrometry.
Luminescence ligand binding methods detect the interaction between a ligand and its binding partner by measuring light emitted during the binding event, offering a sensitive, safe, and often high-throughput alternative to traditional binding assays.
The NanoBiT method detects protein-protein interactions by splitting the NanoLuc luciferase into two inactive subunits (LgBiT and SmBiT) that, when fused to interacting proteins, reconstitute to produce a measurable bioluminescent signal, allowing for sensitive and real-time analysis, especially in live cells.