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Molecular Pathologic Diagnostics for Personalized Medicine
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
ISH is a molecular method used for the detection and localization of specific RNAs or DNAs in cells using tissue sections.5 Through this method, labeled RNA or DNA probes, which are complementary to a target gene sequence, are applied to tissue sections and hybridize with a target gene sequence in the cells. The hybrids can be detected by fluorescent (FISH), silver (SISH), or chromogenic (CISH) signal detection methods. In precision medicine, amplification of HER2, FGFR2, or MET and translocation of ALK are easily, reliably, and accurately detected in FFPE sections using ISH, making ISH the gold standard method (Fig. 6.3).6–9 ISH is also an excellent method for detecting viral infections, such as Epstein–Barr virus latent infection (Fig. 6.4a). In addition, the expression levels of various mRNAs, long noncoding RNAs, and microRNAs within tissue sections can be investigated by RNA ISH (Fig. 6.4b,c).10,11 The ISH procedure consists of pretreatment steps, including proteolysis and acetylation, followed by hybridization of probes in tissue sections and probe signal detection.
Histological approaches
Published in Maxine Lintern, Laboratory Skills for Science and Medicine, 2018
Of course choosing the right probe is very important. Probes can be made from either DNA or RNA, depending on the application and the nature of the target. You can target both DNA and RNA with either type of probe, and each has its own advantages. DNA probes remain stable over long periods of time (months to years), unlike their more labile RNA counterparts. However RNA probes hybridise more efficiently than DNA probes to RNA target sequences.
DNA Analysis
Published in Steven H. Y. Wong, Iraving Sunshine, Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, 2017
Victor W. Weedn, Demris A. Lee, Rhonda K. Roby, Mitchell M. Holland
Sequence information can be obtained by direct sequencing of the DNA locus or more quickly, easily, and inexpensively by probe hybridization. The PCR-based typing technique which is most commonly used at present, is referred to as the reverse dot blot method.49–51 Dot blots involve a series of DNA probes to detect specific target sequences in a given region of DNA. A DNA probe is a small piece of single-stranded DNA (oligonucleotide) which will bind to other single-stranded DNA with the complementary sequence. A sequence-specific oligonucleotide (SSO) probe, also known as an allele-specific oligonucleotide (ASO) probe, is generally composed of 20 to 35 nucleotides. These probes are long enough to confer great specificity, and yet sufficiently short to be destabilized by a single base mismatch, so that it binds only to the exact complement sequence. SSO probes are used to detect the presence or absence of alternative sequence types.
The current and future applications of in situ hybridization technologies in anatomical pathology
Published in Expert Review of Molecular Diagnostics, 2022
Hoi Yi Leung, Martin Ho Yin Yeung, Wai Tung Leung, King Hin Wong, Wai Yan Tang, William Chi Shing Cho, Heong Ting Wong, Hin Fung Tsang, Yin Kwan Evelyn Wong, Xiao Meng Pei, Hennie Yuk Lin Cheng, Amanda Kit Ching Chan, Sze Chuen Cesar Wong
Lin et al. [67] developed amplification-based single-molecule FISH (asmFISH) which is the combination of a modified smFISH with RCA. First, a pair of DNA probes is used to target the RNA molecule of interest. The probe is then ligated to the RNA by enzymatic reaction, forming a circular DNA for signal amplification via RCA. Lastly, fluorescently labeled probes are hybridized to the amplified DNAs and detected by microscopy. To test the applicability of asmFISH to detect gene expression, HER2 expression in two breast cancer cell lines, MCF-7 and SK-BR-3, were measured. The results matched with the human protein atlas database, therefore, asmFISH could show the difference in levels of gene expression. It also had a higher capacity in discriminating SNPs compared to in situ padlock probe assay. The author demonstrated that asmFISH is applicable in both FFPE and fresh frozen tissue samples, showing the potential in clinical application.
Multifaceted applications of pre-mature chromosome condensation in radiation biodosimetry
Published in International Journal of Radiation Biology, 2020
Usha Yadav, Nagesh Nagabhushana Bhat, Kapil Bansidhar Shirsath, Utkarsha Sagar Mungse, Balvinder Kaur Sapra
For the detection of dicentric chromosomes, G0-PCC spreads from irradiated samples were hybridized with centromere specific Peptide Nucleic acid (PNA) probe. Since the centromeric regions are not clearly visible in prematurely condensed chromosomes, FISH with centromeric probe offered an advantage for visualizing dicentric chromosomes. The chromosomal bodies with one, two or no centromeres were clearly visible as shown in Figure 3(d). Besides, excess fragments are not usually considered as radiation specific compared to dicentrics. Hence demand for radiation specific dicentric signal can be met with G0-PCC using PNA-FISH probes even when conventional DCA is not feasible. These probes are more economical than conventionally available DNA probes. The centromeres staining allow quantification of dicentrics. Dicentric based dose estimations are expected to be more accurate as it is well established that dicentrics are most radiation specific markers with low background frequency and low inter-individual variation. Dicentric is known as gold standard for biodosimetry. Dicentric detection using this method is also important for high doses or high LET exposures wherein a big proportion of cells fail to reach metaphases and thus actual dose estimation can be difficult. There are few reports recently been published on dicentric detection with G0-PCC (Karachristou et al. 2015; M’kacher et al. 2015).
Gold nanoparticles and hepatitis B virus
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Babak Negahdari, Mohammad Darvishi, Ali Asghar Saeedi
A number of research works have established that AuNPs based devises can be used to detect HCV. For instance, Griffin et al conceived and developed a procedure known as size and distance dependent nanoparticle surface energy transfer technique for the purpose of HCV RNA sensing and detection [26]. In brief, the underlining principle of the technique shows that RNA probe is branded with fluorophores and absorbed onto the surface of the AuNPs such that once the probe binds to the targeted RNA, the dsRNA complex is released into the solution and a fluorescence emission is reestablished. It is important to note that the resultant solution undergo a color change from red to blue due to aggregation of AuNPs (Figure 1). The fluorescence intensity is directly proportional to the concentration of the target RNA in the solution hence could be utilized in determining the quantity of HCV RNA [26]. In a related development, Glynou et al fabricated a dry-reagent strip biosensor for target DNA detection using AuNPs as visual probe. The results obtained compares favorably with no marked differences between it and COBAS AMPLICORTM, a proven and an efficient kit. Further to it merit, the test is guileless with a reported turnaround time of ∼10 min [27].