Techniques in Quantification and Localization of Gene Expression
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Additionally, processes like covalent additions to amino acid side chain residues can often be reversed by other enzymes. However, some, like the proteolytic cleavage of the protein backbone, are irreversible. PTMs play many important roles in the cell. Measuring gene expression is an important part of many life sciences , as the ability to quantify the level at which a particular gene is expressed within a cell, tissue or organism can provide a lot of valuable information.
For example, measuring gene expression can:. Similarly, the analysis of the location of protein expression is a powerful tool, and this can be done on an organismal or cellular scale. Investigation of localization is particularly important for the study of development in multicellular organisms and as an indicator of protein function in single cells. Ideally, measurement of expression is done by detecting the final gene product for many genes, this is the protein ; however, it is often easier to detect one of the precursors, typically mRNA and to infer gene-expression levels from these measurements.
Levels of mRNA can be quantitatively measured by northern blotting , which provides size and sequence information about the mRNA molecules.
A sample of RNA is separated on an agarose gel and hybridized to a radioactively labeled RNA probe that is complementary to the target sequence. The radiolabeled RNA is then detected by an autoradiograph. Because the use of radioactive reagents makes the procedure time consuming and potentially dangerous, alternative labeling and detection methods, such as digoxigenin and biotin chemistries, have been developed.
Perceived disadvantages of Northern blotting are that large quantities of RNA are required and that quantification may not be completely accurate, as it involves measuring band strength in an image of a gel.
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On the other hand, the additional mRNA size information from the Northern blot allows the discrimination of alternately spliced transcripts. In this technique, reverse transcription is followed by quantitative PCR. The cDNA template is then amplified in the quantitative step, during which the fluorescence emitted by labeled hybridization probes or intercalating dyes changes as the DNA amplification process progresses.
With a carefully constructed standard curve, qPCR can produce an absolute measurement of the number of copies of original mRNA, typically in units of copies per nanolitre of homogenized tissue or copies per cell. For expression profiling , or high-throughput analysis of many genes within a sample, quantitative PCR may be performed for hundreds of genes simultaneously in the case of low-density arrays. A second approach is the hybridization microarray.
A single array or "chip" may contain probes to determine transcript levels for every known gene in the genome of one or more organisms. An advantage of tag-based methods is the "open architecture", allowing for the exact measurement of any transcript, with a known or unknown sequence. Next-generation sequencing NGS such as RNA-Seq is another approach, producing vast quantities of sequence data that can be matched to a reference genome.
Although NGS is comparatively time-consuming, expensive, and resource-intensive, it can identify single-nucleotide polymorphisms , splice-variants, and novel genes, and can also be used to profile expression in organisms for which little or no sequence information is available. Profiles like these are found for almost all proteins listed in Wikipedia. Additional information can be found by searching their databases for an example of the GLUT4 transporter pictured here, see citation. For genes encoding proteins, the expression level can be directly assessed by a number of methods with some clear analogies to the techniques for mRNA quantification.
The most commonly used [ citation needed ] method is to perform a Western blot against the protein of interest—this gives information on the size of the protein in addition to its identity. A sample often cellular lysate is separated on a polyacrylamide gel , transferred to a membrane and then probed with an antibody to the protein of interest.
The gel-based nature of this assay makes quantification less accurate, but it has the advantage of being able to identify later modifications to the protein, for example proteolysis or ubiquitination, from changes in size. Quantification of protein and mRNA permits a correlation of the two levels.
The question of how well protein levels correlate with their corresponding transcript levels is highly debated and depends on multiple factors. Regulation on each step of gene expression can impact the correlation, as shown for regulation of translation  or protein stability. Analysis of expression is not limited to quantification; localisation can also be determined. The probed sample is then observed by microscopy to identify where the mRNA or protein is.
By replacing the gene with a new version fused to a green fluorescent protein or similar marker, expression may be directly quantified in live cells. This is done by imaging using a fluorescence microscope. It is very difficult to clone a GFP-fused protein into its native location in the genome without affecting expression levels so this method often cannot be used to measure endogenous gene expression. It is, however, widely used to measure the expression of a gene artificially introduced into the cell, for example via an expression vector.
It is important to note that by fusing a target protein to a fluorescent reporter the protein's behavior, including its cellular localization and expression level, can be significantly changed. The enzyme-linked immunosorbent assay works by using antibodies immobilised on a microtiter plate to capture proteins of interest from samples added to the well.
Using a detection antibody conjugated to an enzyme or fluorophore the quantity of bound protein can be accurately measured by fluorometric or colourimetric detection. The detection process is very similar to that of a Western blot, but by avoiding the gel steps more accurate quantification can be achieved.
An expression system is a system specifically designed for the production of a gene product of choice. In the broadest sense this includes every living cell but the term is more normally used to refer to expression as a laboratory tool.
An expression system is therefore often artificial in some manner. Expression systems are, however, a fundamentally natural process. Viruses are an excellent example where they replicate by using the host cell as an expression system for the viral proteins and genome. Doxycycline is also used in "Tet-on" and "Tet-off" tetracycline controlled transcriptional activation to regulate transgene expression in organisms and cell cultures.
In addition to these biological tools, certain naturally observed configurations of DNA genes, promoters, enhancers, repressors and the associated machinery itself are referred to as an expression system. This term is normally used in the case where a gene or set of genes is switched on under well defined conditions, for example, the simple repressor switch expression system in Lambda phage and the lac operator system in bacteria.
Several natural expression systems are directly used or modified and used for artificial expression systems such as the Tet-on and Tet-off expression system.
Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as transcription factors , and outputs being the level of gene expression. The node itself performs a function, and the operation of these functions have been interpreted as performing a kind of information processing within cells and determines cellular behavior. Gene networks can also be constructed without formulating an explicit causal model. This is often the case when assembling networks from large expression data sets. The source of variation can be either experimental or natural observational.
There are several ways to construct gene expression networks, but one common approach is to compute a matrix of all pair-wise correlations of expression across conditions, time points, or individuals and convert the matrix after thresholding at some cut-off value into a graphical representation in which nodes represent genes, transcripts, or proteins and edges connecting these nodes represent the strength of association see .
The following experimental techniques are used to measure gene expression and are listed in roughly chronological order, starting with the older, more established technologies. They are divided into two groups based on their degree of multiplexity. From Wikipedia, the free encyclopedia.
For vocabulary, see Glossary of gene expression terms. For a non-technical introduction to the topic, see Introduction to genetics. Conversion of a gene's sequence into a mature gene product or products. Main article: Transcription biology. Main article: Post-transcriptional modification.
Main article: Translation genetics.
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Main article: Protein folding. Main article: Regulation of gene expression.
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Main article: Transcriptional regulation. Main article: Regulation of transcription in cancer. Main article: Post-transcriptional regulation. Main article: Three prime untranslated region. Main article: MicroRNA. Main article: Post-translational modification. Main articles: In situ hybridization and Immunofluorescence. Main article: Protein production biotechnology. Main article: Gene regulatory network. AlloMap molecular expression testing Bookmarking Expressed sequence tag Expression Atlas Expression profiling Gene structure Genetic engineering Genetically modified organism List of biological databases List of human genes Oscillating gene Paramutation Protein production Protein purification Ribonomics Ridge Sequence profiling tool Transcriptional bursting Transcriptional noise Transcript of unknown function.
Moreover, using strain-promoted cycloaddn. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous. Starck, Shelley R. Understanding the expression of known and unknown gene products represents one of the key challenges in the post-genomic world.
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Here, we have developed a new class of reagents to examine protein expression in vivo that does not require transfection, radiolabeling, or the prior choice of a candidate gene. To do this, we constructed a series of puromycin conjugates bearing various fluorescent and biotin moieties. These compds. Overall, this work demonstrates that fluorescent-puromycin conjugates offer a general means to examine protein expression in vivo. David, Alexandre; Dolan, Brian P. Whether protein translation occurs in the nucleus is contentious.
To address this question, we developed the ribopuromycylation method RPM , which visualizes translation in cells via std. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin PMY -specific monoclonal antibody in fixed and permeabilized cells.
The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chem.