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Enteric Pathogens
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
A riboprobe derived from a cDNA fragment of a 650-bp HDV RNA sequence has been developed for spot hybridization. Its advantages are: it can be used on large batches of specimens more easily than the northern blot hybridization assay using a cDNA of HDV RNAresults of riboprobe spot hybridization correlate well with those of northern blottingthe riboprobe assay offers a substantial enhancement in sensitivity (83 vs. 63%)it offers significant savings in time compared to the northern blot assay using a cDNA probewhen detecting HDV viremia, the riboprobe method proved to be the most sensitive of the hybridization-based methods described so far
The Luteinizing Hormone–Releasing Hormone System in the Developing Monkey Brain
Published in Akira Matsumoto, Sexual Differentiation of the Brain, 2017
Ei Terasawa, Laurie A. Abler, Nancy M. Sherwood
In situ hybridization histochemistry with monkey and rat mLHRH cRNA riboprobes suggests that the distribution of late cells expressing mLHRH mRNA (Figure 16.2a) is essentially identical to those stained with GF-6 (Figure 16.2b). The results clearly indicate that late LHRH cells originating from the olfactory pit express mLHRH mRNA as expected. This observation is in agreement with those described by Wray et al.31 and Ronnekleiv and Resko.3 Early LHRH cells in the basal forebrain of young fetuses (E35 to E39) and in the striatum and amygdala in older fetuses (E50 to E78) also express mLHRH mRNA. The distribution of mLHRH mRNA positive early cells (Figure 16.2c) in the forebrain is very similar to that seen with GF-6 (Figure 16.2d). However, the expression of mLHRH mRNA in early cells is consistently less than that seen in late cells, suggesting that early cells contain low transcription levels of the mLHRH gene. A double-labeling study with GF-6 and antisense digoxigenin-labeled riboprobes suggest that both late and early LHRH neurons express mLHRH mRNA. In contrast, in situ hybridization using sense riboprobe does not hybridize with mLHRH mRNA. In addition, we have confirmed the specificity of the antisense riboprobe hybridization with early and late cells using a “random” digoxigenin-labeled riboprobe, as well as prehybridizing the sections with an excess of unlabeled antisense riboprobe to block antisense hybridization.
In Situ Hybridization
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
The choice between these two methods is largely based on personal preference, although separate denaturation is most commonly used in molecular cytogenetics for fluorescent in situ hybridization (FISH); co-denaturation is most commonly used to analyze tissue sections.8–15 The main advantage of co-denaturation is the reduction of the number of practical steps. However, some argue that morphological preservation is less optimal than with separate probe and target denaturation. Riboprobes and oligonucleotide probes are single-stranded, as is cellular RNA. Therefore, denaturation is not essential for RNA detection but improves the sensitivity of riboprobe detection of RNA, possibly by removing the secondary structure of RNA probe and target.
Adrenal-dependent and -independent stress-induced Per1 mRNA in hypothalamic paraventricular nucleus and prefrontal cortex of male and female rats
Published in Stress, 2018
Lauren E. Chun, Jenny Christensen, Elizabeth R. Woodruff, Sarah J. Morton, Laura R. Hinds, Robert L. Spencer
In situ hybridization for Per1, Per2, Bmal1, and cFos mRNA followed procedures previously reported (Chun et al., 2015). Briefly, sections on slides were fixed with 4% paraformaldehyde, went through a series of standard saline citrate (SSC) solution washes, then bathed in triethanolamine and acetic anhydride solution. Sections were dehydrated in increasing concentrations of ethanol baths before being air dried. Hybridization buffer containing the 35S-labelled riboprobe for each gene of interest was applied to each slide. Hybridization occurred in humidified chambers with 50% formamide and 50% water for 16–20 h. Coverslips were gently removed in SSC baths. Tissue was then exposed to 0.02 g/L RNase at 37 °C, washed in decreasing concentrations of SSC, and incubated in SSC at 65 °C for 1 h. Sections were dehydrated in increasing concentrations of ethanol, then air dried. Slides were set on Kodak BioFilm Maximum Resolution Autoradiography Film (Carestream Health, Windsor, CO) for 2–4 weeks, then developed in a medical film processor SRX-101A (Konica Minolta, Tokyo, Japan). Each in situ hybridization assay was separated by experiment and by region of interest (ROI; SCN and PVN were run together).
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
There are a variety of probes available for ISH. Complementary DNA (cDNA) and RNA probes (riboprobes) are made from cloning using a cloned nucleic acid sequence inserted into a plasmid vector. It is simpler to label by nick translation for cDNA probes but they must be denatured before applying to the tissues. Riboprobe is a product from the probe cDNA template by ribonucleic acid polymerase. Oligonucleotide probes are synthetic oligonucleotide DNA probes with the length of 20 to 50 bases [16].
Modulation of neuromuscular synapses and contraction in Drosophila 3rd instar larvae
Published in Journal of Neurogenetics, 2018
Kiel G. Ormerod, JaeHwan Jung, A. Joffre Mercier
Proctolin was the first insect neuropeptide identified (Brown & Starratt, 1975) and is broadly conserved across the arthropods (Christie, 2014, 2015; Christie et al., 2010; Isaac, Taylor, Hamasaka, Nässel, & Shirras, 2004; Orchard, Belanger, & Lange, 1989) but appears to be absent from a few insect species (Caers et al., 2012). Proctolin acts as a co-transmitter and neurohormone and modulates neuromuscular synapses, muscle contraction, cardiac contraction, circulation, stomach and gut motility, reproductive organs and sensory coding (Adams & O’Shea, 1983; Erxleben, deSantis, & Rathmayer, 1995; Kravitz et al., 1980; Lange, 2002; Marder, Hooper, & Siwicki, 1986; Orchard et al., 1989; Pasztor & MacMillan, 1990). The first gene encoding a proctolin precursor protein was identified in Drosophila (Taylor et al., 2004). Evidence for the presence of the proctolin peptide (RYLPT) in Drosophila is based on: (a) chromatographic analysis of CNS and tissue extracts with subsequent testing using bioassay and immunoassay (Anderson et al., 1988), (b) immunohistochemical staining using antibodies against proctolin or the proctolin precursor (Anderson et al., 1988; Taylor et al., 2004), (c) in situ hybridization with a riboprobe to the Proct gene (Taylor et al., 2004) and (d) the presence of proctolin in clones derived from a cell line of Drosophila CNS (Ui-Tei, Sakuma, Watanabe, Miyake, & Miyata, 1995). Extraction and sequencing of the authentic peptide from Drosophila, however, have not been achieved. We recently subjected an extract from ten Drosophila 3rd instar larvae to matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and our results (Figure 2) show a very small peak corresponding to a molecular mass (649.6 Da) close to that predicted for proctolin (648 Da). We do not know why recovery of substantial amounts of proctolin from Drosophila extracts is so difficult.