Course: SAT 170 Introductory Biotechnology

Unit 7 Reading and Multimedia Assignment

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“Techniques to study expression of a single gene” by Boundless, CC BY SA 3.0

https://www.boundless.com/biology/biotechnology/biotechnology-reveals-gene-sequence-expression-and-function/techniques-to-study-expression-single-gene/

Reading Assignment: Techniques to study expression of a single gene
Northern blotting, RT-PCR, and in situ hybridization (ISH) are used to study gene expression by detecting RNA or mRNA in a tissue.

PCR and Electrophoresis

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part or all of the target sequence. This technique is in contrast to Southern blotting, which separates and identifies DNA sequences, and Western blotting, which does the same for proteins. The term "northern blot" actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However, the entire process is commonly referred to as "northern blotting."

A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. The hybrid signals are then detected by X-ray film and can be quantified by densitometry.

Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment. The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as gene expression in the rejection of transplanted organs. If an upregulated gene is observed by an abundance of mRNA on the northern blot, the sample can then be sequenced to determine if the gene is known to researchers or if it is a novel finding. The expression patterns obtained under given conditions can provide insight into the function of that gene. For example, the expression of the B-globulin gene at different stages of development in the hummingbird can be compared by northern blotting.

A more modern technique for analyzing gene expression is reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is a variant of polymerase chain reaction (PCR), a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed amplification. In RT-PCR, however, an 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 PCR or real-time PCR. The analysis of the PCR products depends on the type of PCR applied. If a conventional PCR is used, the PCR product is detected using agarose gel electrophoresis and ethidium bromide (or other nucleic acid staining) (see above figure).

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH). RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts, another way to determine gene expression.