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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
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. 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. A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe—RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation. 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. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound specifically and to avoid background signals from arising. The hybrid signals are then detected by x-ray film and can be quantified by densitometry (Figure 3.14).
Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Northern blotting is similar, except the gel must be denaturing as discussed previously, because the target is RNA. As in Southern blotting, the probes may be DNA or RNA. (The use of an RNA probe to hybridize to DNA is sometimes also done and is called a reverse Northern blot.) Although rt-PCR has largely replaced Northern blotting in medicine, most molecular biologists consider the Northern blot the gold standard for the direct measurement of mRNA expression levels. It is also used to study RNA degradation, half-lives, and alternative splicing.
Iron oxide/poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) glassy carbon electrode as a novel label-free electrochemical microRNA-21 sensor
Published in Instrumentation Science & Technology, 2023
Cigdem Dulgerbaki, Aysegul Uygun Oksuz
Today, miRNA detection methods are usually based upon the gold standard technique; Northern blot; the quantitative reverse transcription polymerase chain reaction (qRT-PCR); RNA-sequencing, and microarrays.[9,10] These techniques exhibit excellent analytical performance; however, they are complicated, expensive and include long detection procedures. For this reason, they are not convenient for resource-limited facilities limiting their implementation. In recent years, growing attention has been developed for building diverse biosensing strategies that enable the quantification of miRNAs sensitively and selectively.[11]