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Molecular biology
Published in Maxine Lintern, Laboratory Skills for Science and Medicine, 2018
Northern blotting is often used to discover if the RNA for a particular gene is present in a particular cell. DNA may well be the master copy of the information to make an organism, but unless a particular gene is being expressed (first by making RNA, and then ultimately the protein for which that gene codes), then the cell isn’t ‘using’ that gene. Measuring the levels of RNA for a specific gene can give you a good indication of the level of expression of that gene, and a possible hint at the potential protein levels. Unlike DNA, which is pretty tough, RNA is a much more fragile, heat labile, fall apart as soon as you look at it kind of molecule. Thus much of the detail of the northern method is concerned with preserving the RNA as much as possible, so as to result in a representative value for the RNA levels in a particular cell.
Molecular Biology
Published in John C Watkinson, Raymond W Clarke, Louise Jayne Clark, Adam J Donne, R James A England, Hisham M Mehanna, Gerald William McGarry, Sean Carrie, Basic Sciences Endocrine Surgery Rhinology, 2018
Michael Kuo, Richard M. Irving, Eric K. Parkinson
Hybridization is the specific annealing of single DNA (or RNA) strands, the probe, to a DNA sample, the target. It serves to detect the presence of a specific sequence of DNA either in the cell or on a hybridization membrane and recognition that hybridization has occurred is achieved either by radioactively labelling the probe and localizing the radioactivity by autoradiography or by labelling the probe with fluorochromes that fluoresce when excited by light of specific wavelengths (Figure 1.2). Hybridization on a membrane requires the initial transfer of DNA on to a nitrocellulose membrane from an agarose gel. This elegantly simple process is eponymously known as Southern blotting after the scientist who described the process in 1975. Two other commonly used transfer techniques have their names derived from Southern blotting as jargon terms. Northern blotting is essentially the same process used for transfer of RNA to a membrane. Western blotting is one of the mainstays of protein analysis and involves the transfer of electrophoresed protein bands from a polyacrylamide gel on to a nitrocellulose or nylon membrane to which they bind strongly. Detection of the protein is usually achieved by the use of antibodies to specific antigens presented by the protein with the antibody being labelled radioactively, enzymatically or fluorescently.
SBA Answers and Explanations
Published in Vivian A. Elwell, Jonathan M. Fishman, Rajat Chowdhury, SBAs for the MRCS Part A, 2018
Vivian A. Elwell, Jonathan M. Fishman, Rajat Chowdhury
The polymerase chain reaction (PCR) is an amplification process used to amplify small amounts of DNA in order to perform analysis. It does not identify specific sequences. The DNA can then be analysed using Southern blotting. PCR involves synthesizing two oligonucleotide primers, that is short segments of RNA, that will bind to the DNA and when added to denatured DNA will bind to the DNA and amplify the DNA. The cycle is continually repeated 20–30 times, resulting in an exponential increase in the quantity of DNA. Reverse transcription PCR uses RNA. RNA is too unstable to be used for PCR, so it must be converted to a complementary copy of DNA using reverse transcriptase. PCR is then performed. Southern blotting involves digestion of DNA and are denatured in alkali making them single-stranded. A permanent copy of the single strands is made by placing the DNA on a nitrocellulose filter – that is, the Southern blot. A target radioactively labelled DNA fragment is then added and will bind to its homologous DNA fragment (if present). The DNA is then washed to remove any unbound DNA. The hybridized DNA can then be visualized as a band using autoradiography.Northern blotting is similar to Southern blotting but uses mRNA as the target nucleic acid, rather than DNA. The mRNA can be hybridized to a radiolabelled DNA probe.Western blotting is used to analyse proteins that are separated by electrophoresis, transferred to nitrocellulose, and reacted with antibody for detection.
Overview of gene expression techniques with an emphasis on vitamin D related studies
Published in Current Medical Research and Opinion, 2023
Jeffrey Justin Margret, Sushil K. Jain
Northern blotting, one of the first techniques used to analyze a sample of RNA from a specific cell type or tissue, measures the RNA expression of distinct genes. The expression profile of the gene determined under certain conditions can provide insight into its function7. This relatively laborious method relies on the hybridization between a known nucleic acid probe and the complementary sequence in the mixture of RNAs8. The binding of the probe to the membrane confirms the presence of a complementary RNA sequence in a given sample (Figure 2). These RNA-probe complexes can be detected using different radionuclide labeling. Since this technique uses size-dependent separation, it can only determine the abundance of selected RNA and the size of the transcript9. Northern blotting is a variant of Southern blotting, which is used to analyze DNA, and their protocols are similar. It can also be very effective at detecting novel splice site variants, characterization, and verification of small RNAs, such as miRNAs8. Although its relatively high specificity reduces false positives10, the sensitivity of the Northern blot is diminished if the amount of total RNA or the expression level of the transcript is low9. Recently, the use of Northern blotting in research has been limited due to its low sensitivity, visualization of one or only a few genes at a time, and degradation of the samples by RNases.