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Introduction to Cells, DNA, and Viruses
Published in Patricia G. Melloy, Viruses and Society, 2023
Virologists are scientists who study viruses. They typically conduct their work in laboratory settings, although they occasionally conduct their research outside the laboratory and engage in more field work. A virologist might conduct experiments using live animal models (in vivo research) or work with mammalian or other animal cells grown in culture in the laboratory (in vitro research). One commonly used technique to study viral growth in the lab is known as a plaque assay. In this technique, host cells are added to media in a petri dish, and then the cells are infected with virus for several days. Scientists then monitor the clearings in the cell layer created by the virus killing cells in that local area. The clearing is known as a plaque. The number of plaques can be counted to quantify the amount of virus present (Lostroh 2019). The virologist uses all the tools that a typical cell and molecular biologist might use to analyze genes and gene expression into a protein. DNA sequencing is used to compare one virus to another. A powerful technique used to amplify DNA sequences is known as polymerase chain reaction (PCR), and this tool can be used for medical diagnostics to look for viral nucleic acid as well (Nobel Media AB 1993). Techniques such as northern blot and western blot are used to study RNA and protein expression respectively. Scientists may also use online databases or tools to study viral DNA sequences or protein structure.
Transcriptional Regulation of the Human C3 Gene
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Gretchen J. Darlington, Deborah R. Wilson, Todd S.-C. Juan
Northern-blot analysis was performed by standard procedures.30 Lanes contained 20 μg of total cellular RNA. Probes used in hybridization were inserts isolated from the human albumin cDNA clone, F47,31 the human C3 cDNA clone, pHC3.11,32 the human α-fibrinogen cDNA clone, p115.6,33 and the human γ-fibrinogen cDNA clone, p253.34
Laboratory techniques to study the cellular and molecular processes of disorders
Published in Louis-Philippe Boulet, Applied Respiratory Pathophysiology, 2017
Northern blot is used to detect a specific messenger RNA (mRNA) in a mixture of RNA sample. It is based on the same principles as Southern blot, except that the RNA strands in Northern blot do not need to be fragmented by a restriction enzyme [16].
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.
miRNAs as attractive diagnostic and therapeutic targets for Familial Mediterranean Fever
Published in Modern Rheumatology, 2021
Hamza Malik Okuyan, Mehmet A. Begen
Northern Blot Hybridization is a laborious, time-consuming and low-sensitivity method for miRNAs detection. The method includes gel electrophoresis, membrane transfer, cross-linking and probe hybridization steps [102,103]. Next-generation sequencing (NGS) is an advanced technique that enables the quantification of known miRNA and the identification of novel miRNAs. Currently, the use of NGS is limited in clinical settings due to its high cost and bioinformatics analysis requirements [101,104]. Microarrays, based on the nucleic acid hybridization method, are one of the most frequently used methods with low-cost for expression profiling of miRNAs. Microarrays enable the profiling of a large number of miRNAs in a single operation and preferably can be used to compare healthy and patients groups. However, it usually requires validation by another method such as qRT-PCR due to restricted specificity caused by cross-hybridization during microarray [80,101,102]. qRT-PCR is a highly sensitive technique, considered as the gold standard for quantification of gene expression and widely used in genetic laboratories [101]. This method allows the detection of a very small alteration of miRNAs expressions thanks to its high sensitivity, specificity, reproducibility, and accuracy [80]. Also, widely available commercial ready to use kits simplify the use of this technique. The detection of miRNA expression by qRT-PCR includes a reverse transcription of miRNA to cDNA, then, amplification of the target gene with qPCR [102].
The effect of Jiedu Huoxue decoction on rat model of experimental nonbacterial prostatitis via regulation of miRNAs
Published in Pharmaceutical Biology, 2020
Zhangren Yan, Chunhua Huang, Gang Huang, Yunbo Wu, Jiangang Wang, Jun Yi, Wenli Mao, Wanchun Wang
According to the differential expression profiles obtained by sequencing, all the miRNAs expressed in the two groups of samples were calculated. We used edgeR to determine whether there were significant differences between the two groups of samples. There were 211 known miRNAs with significant differences between the normal control group and model group. According to the predicted results of target genes, 37 were prostate-related, and 181 were differentiated miRNAs between the model group and high dose JDHXD group, of which 23 intersected with the differentiated molecules of the normal control group and model group. We selected four molecules (miR-146a, miR-96, miR-423, miR-205) for further validation. As shown in Figure 8(A), the results of qRT-PCR validation were consistent with the sequencing trend. The expressions of miR-146a, miR-423 and miR-205 increased significantly in the model group (p < 0.05). After drug treatment, they showed a downward trend, and a dose dependence trend was noted in the Chinese medicine group. The expression of miR-96 decreased significantly in the model group (p < 0.05) and showed a recovery trend after drug intervention. Figure 8(B) is the exposure map and statistical analysis results of the Northern blot. The trends of miR-146a, miR-96, miR-423 and miR-205 were consistent with the results by qRT-PCR. These results suggested that drug intervention can induce significant changes in molecules in the model group towards normal levels and these miRNAs are likely to participate in the development of CAP/CPPS.