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An oncolytic virus is considered a targeted cancer therapy due to its ability to specifically target, replicate in and lyse cancer cells while leaving normal cells unharmed. Over the last few years several tumor selective oncolytic viruses have been developed. These include certain DNA viruses such as adenovirus that have been genetically manipulated to target specific cancer cells by exerting restrictions...
This manuscript details a protocol for the co-localization of a microRNA and its putative protein target in paraffin embedded formalin fixed tissues. The key variables for the first step, microRNA in situ hybridization, includes probe concentration (1–2pmol/μl), locked nucleic acid (LNA) modified probes, protease digestion (pepsin 1.3mg/ml), and a low stringency wash. Key variables for the subsequent...
Zinc (Zn) is essential for a very large number and variety of cellular functions but is also potentially toxic. Zn homeostasis is therefore dynamically maintained by a variety of transporters and other proteins distributed in distinct cellular and subcellular compartments. Zn transport is mediated by two major protein families: the Zip family, which mediates Zn influx, and the ZnTs which are primarily...
With the advent of personalized medicine, anatomic pathology-based molecular assays, including in situ hybridization (ISH) and mRNA detection tests, are performed routinely in many laboratories and have increased in their clinical importance and complexity. These assays require appropriately fixed tissue samples that preserve both nucleic acid targets and histomorphology to ensure reliable test results...
Epigenetics in general and microRNA (miRNA) in particular are an important and growing field of research, and while significant advances in the role of miRNA in a variety of diseases including cancer have been made, the majority of the information on the relationship between miRNA and its putative target proteins have been made on homogenized tissue, which, while useful, provides no information on...
Central nervous system consists of a myriad of cell types. In particular, many subtypes of neuronal cells, which are interconnected with each other, form the basis of functional circuits. With the advent of genomic era, there have been systematic efforts to map gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy. To make full use of such information, it is important...
The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by...
Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and...
Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization...
The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent...
Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given...
MicroRNAs (miRNAs) constitute a group of small non-coding RNA molecules generally 18–22 base-pairs in length. miRNAs are considered to be negative regulators of gene expression at the level of post-transcription and are important in normal physiological development, tissue homeostasis and disease. The cellular origin of individual microRNAs is of utmost importance for understanding their roles in...
This manuscript focuses on the detection of viral nucleic acids by in situ based methodologies. The optimal protocol depends on the virus. We will describe protocols for viral RNA detection by reverse transcriptase (RT) in situ PCR. We will also directly compare this method to the detection of viral RNA using standard in situ hybridization with locked nucleic acid (LNA) probes. Most DNA viruses are...
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