Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot. RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise. They can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence. Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest. Since the large ribosomal subunit is 28S (approximately 5kb) and the small ribosomal subunit is 18S (approximately 2kb) two prominent bands appear on the gel, the larger at close to twice the intensity of the smaller. An RNA ladder is often run alongside the samples on an electrophoresis gel to observe the size of fragments obtained but in total RNA samples the ribosomal subunits can act as size markers. Polyacrylamide gel electrophoresis with urea can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs. The gels can be stained with ethidium bromide (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting. The RNA samples are most commonly separated on agarose gels containing formaldehyde as a denaturing agent for the RNA to limit secondary structure. Gels RNA run on a formaldehyde agarose gel to highlight the 28S (top band) and 18S (lower band) ribosomal subunits. To create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by microarrays or RT-PCR. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. The membrane is washed to ensure that the probe has bound specifically and to prevent background signals from arising. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. After a probe has been labeled, it is hybridized to the RNA on the membrane. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. 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. Capillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane.Ī 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. 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. RNA samples are then separated by gel electrophoresis. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail. Procedure Ī general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. However, the entire process is commonly referred to as northern blotting. Strictly speaking, the term 'northern blot' refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression rates during differentiation and morphogenesis, as well as in abnormal or diseased conditions. The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. Molecular biology technique Flow diagram outlining the general procedure for RNA detection by northern blotting.
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