Microscopy and related techniques
C M Langton, C F Njeh in The Physical Measurement of Bone, 2016
This is mentioned briefly because similar principles of intermolecular affinity to those above apply. In situ hybridization is used by molecular biologists as a research tool at the interface of biochemistry and histology for the location and expression of nucleic acids within histological sections, an advantage being that the isolation and purification of the genes from the tissue is not necessary. Labelled RNA or DNA probes (most commonly cDNA and oligonucleotides, 20–50 mers, the sequence of which is specified) are placed in solution upon fixed histological sections where they locate the complementary nucleic acid chain by forming a specific hybrid molecule that may be DNA + RNA, DNA + DNA or RNA + RNA. Before commencement the hydrogen bonding between the in situ DNA strands are first broken to permit hybridization with the labelled complementary strands of either DNA or RNA. To enable visualization under the optical microscope there is a further (indirect) affinity reaction using a second labelling reagent (table 7.6). This may contain radioactive molecules (for example 35S [8]) for detection by autoradiography (p. 228), or more recently biotin or digoxigenin followed by labelled streptavidin or anti-digoxygenin respectively, or an immunohistochemical reaction including immuno-gold if electron microscopy is proposed (see Bendayan [16] for further general details and Saito et al [31], Kaneko et al [32] and Sherwin et al [33] for application to calcified tissues).
In situ Hybridization Histochemistry
Edythe D. London in Imaging Drug Action in the Brain, 2017
Single-stranded mRNAs are efficiently digested by RNase A, even in fixed tissue sections. Incubation of tissue with RNase prior to hybridization with cRNA probes, therefore, eliminates hybridization signals utilizing either RNA or DNA probes. Any signal persisting in the face of rigorous RNase pretreatment is suspect and is probably caused by nonspecific binding of radioactive molecules to tissue sections. It should be noted that when using single-stranded cRNA probes, RNase treatment should be followed by proteinase K treatment prior to hybridization, as residual RNase may cause breakdown of the applied cRNA probe and prevent occurrence of any nonspecific binding. Pretreatment with RNase is not to be confused with post-hybridization RNase treatment, which digests nonhybridized single-stranded RNAs in the tissue section and serves to minimize background signal.
DNA Analysis
Steven H. Y. Wong, Iraving Sunshine in Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, 2017
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.
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].
A new method for early detection of pancreatic cancer biomarkers: detection of microRNAs by nanochannels
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Jiasheng Xu, Kaili Liao, Zhonghua Fu, Zhenfang Xiong
In this study, we designed a DNA probe (Probe) that can specifically recognize the target miRNA. The probe is fully complementary to the target recognition region of the target miRNA and can be used for specific detection of miRNA. The recognition region of the probe with the miRNA ends as the guide strand enables the αHL to rapidly capture the miRNA·probe molecule [28–32]. The main purpose of this study is to achieve high-efficiency detection of pancreatic cancer miRNAs using the high-sensitivity resolution of miRNA·probe complex molecules by αHL single nanochannel. The experimental results show that due to the difference of the probes sequence, the miRNA 21·probe 21, miRNA155·probe 155, miRNA 196a·probe 196a, three complex molecules form different characteristic shapes and different blocking time characteristic current signals, so the characteristic signal can be used effectively distinguish between miRNA 21, miRNA 155 and miRNA 196a.
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