MCAT Content / Recombinant Dna And Biotechnology / Analyzing Gene Expression

Analyzing gene expression

Topic: Recombinant Dna And Biotechnology

Gene expression is the process by which information from a gene is used to be converted into a functional product, such as a protein, this can be analysed by using a variety of methods.

A number of techniques can be used to measure gene expression, including in situ hybridization, immunohistochemistry, flow cytometry and western blotting.

In Situ Hybridization

In Situ Hybridization is used to study gene expression in tissue or in an embryo. In order to analyze gene expression a thin slice of tissue is fixed to a slide and permeabilized to open the cell membranes. A labeled probe is added to the selection of tissue and it binds to the transcript of interest. An enzyme-linked antibody is then added to the selection of tissue and it binds to the probe. A substrate for the enzyme is then added and the transcript-probe-antibody complex is detected by the substrate. This process is used to detect where transcripts are expressed.

Immunohistochemistry

Immunohistochemistry uses an antibody to detect a specific protein and measure its expression. The antibody used in this process is recognized by a second antibody. The second antibody is linked to either an enzyme or a fluorescent molecule. Immunohistochemistry is often used in clinical studies to detect for certain diseases such as breast cancer. The HER2 gene is targeted in breast tissue biopsies to see if it is highly expressed.

Flow Cytometry

In flow cytometry single cells are either from lab cell culture or tissue samples and are stained for certain protein markers using specific antibodies. The antibodies have a fluorescent tagged to them that are detected using flow cytometry. The labeled cells are suspended in a stream and are passed through a beam of light. As the labeled cells pass through the laser, they emit light. The emitted light is measured giving information on cell size and how many of the cells express the markers that were labeled. Flow cytometers have many different light channels that can be used to detect many different antibodies so many different antibodies can be in one sample all at once. The cells can also be sorted based on the different markers detected by the laser.

Western Blots

Western blots also use protein-specific antibodies to recognize proteins of interest. Gel electrophoresis is used to separate proteins onto a gel, and then the proteins are transferred onto a solid support matrix. This solid matrix is then exposed to antibodies specific to the protein of interest, which can be visualized via fluorescence or an enzymatic reaction. Antibodies can detect specific proteins, and also proteins with specific post-translational modifications like phosphorylation.

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Key Points

• In addition to RT-PCR, qPCR, and western blotting; the three different techniques used to analyze gene expression are in situ hybridization, immunohistochemistry, and flow cytometry

• In situ hybridization uses a labeled gene or modified nucleic acids strand to target a specific gene and analyze its expression

• Immunohistochemistry is a method that uses antibodies to check for certain markers in a sample of tissue

• Flow cytometry is a laser-based technology to analyze the characteristics of cells

• Western blotting uses antibodies to recognize proteins that have been separated by gel electrophoresis and transferred to a membrane


Key Terms

labeled probe: a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome

antibody: a protein that responds to a counteracting specific antigen

in situ hybridization: uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue

immunohistochemistry: the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

western blots: use antibodies to recognize proteins that have been separated by gel electrophoresis and transferred to a membrane



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