Comparison of different labeling strategies for in situ hybridization ranked according to their relative sensitivity.īased On 35S-Labelled Riboprobes And Frozen Sections Over the past 10 years we have compared most of these methods in my laboratory and have come to the conclusion that 35S riboprobes represent the most sensitive method for the detection of mRNA in tissue sections (Table I). There are also multiple ways of labeling these probes using radioactive (3H, 32P, 33P, 35S) or non-radioactive (biotin, alkaline phosphatase, digoxigenin, fluorescent) nucleotides. There are many different ways to do in situ hybridization including probing with cDNAs, cRNAs, or synthetic oligonucleotides. The repeated hybridization of these tissues with multiple probes over a long period of time also allows for the eventual serial reconstruction of a tissue with all of the information concerning mRNA content, cell identity, and regional localization available for study. It is possible therefore to make libraries of tissues, stored as sections in the freezer that can be probed in the future as new probes become available or new questions arise. Kept at -70☌ with desiccant, these tissue sections can be stored for more than six years without significant loss of the hybridization signal (Wilcox, unpublished observations). A single surgical biopsy of a human atherosclerotic plaque coming from carotid endarterectomy surgery, for example, might yield up to 1000 tissue sections each of which can be used for a separate hybridization. However, literally hundreds of different hybridizations can be performed on the same piece of tissue using situ hybridization. While Northern blots can be probed multiple times for different mRNAs, in fact this may be limited to four or five different probes at most, as the transfer membranes break down and there can be some RNA loss with repeated stripping of the blots. These Northern blots would only yield information regarding the presence or absence of mRNAs without any other information as to the cellular source of that RNA. A tissue digest from a surgical biopsy might yield sufficient RNA for one or two Northern blots. clinical biopsies, embryos, cultured cells). Thus, potentially important interactions between cells that express different proteins may be uncovered.Ī major advantage of in situ hybridization is that it allows the maximum use of tissues that may be in short supply (i.e. Furthermore, since in situ hybridization is a histological technique cellular relationships are maintained and it is possible to precisely identify cell types expressing the gene of interest. However, in situ hybridization is exquisitely sensitive and can detect the amount of mRNA contained in a single cell. All of the cellular relationships are lost with Northern blots and mRNA levels are averaged from all of the cells contained in the original sample. The two techniques differ in that the starting material for a Northern blot is a tissue digest while the primary material for in situ hybridization is a histological tissue section. In situ hybridization is very similar to Northern blots and depends on the hybridization of a labeled nucleic acid probe (RNA or DNA) to a complementary sequence of mRNA. A major advance in the method was achieved with the description in 1984 of in situ hybridization using single stranded RNA probes (Cox et al 1984a) otherwise known as "riboprobes". Subsequently, papers appeared in which the technique was used to localize mRNAs for globin (Harrison et al 1973 Conkie et al 1974), visna virus (Brahic, Haase, 1978), growth hormone (Hudson et al 1981), beta-endorphin/ACTH (Hudson et al 1981 Gee et al 1983 Gee, Roberts, 1983) and prolactin (Pochet et al 1981). The technique was first described as a method for the localization of DNA:RNA hybrids in cytological preparations by Gall and Pardue in 1969 (Gall, Pardue, 1969). In situ hybridization is an invaluable tool for the examination of gene expression. Transcription of Digoxigenin-labeled Riboprobes Non-radioactive In Situ Using Digoxigenin-labeled Riboprobesġ1. Solutions for Non-radioactive In Situ (biotinylated)ġ0. Sample Calculations for Non-radioactive In Situĩ. Non-radioactive In Situ Using Biotin-labeled RiboprobesĨ. Labeling of Synthetic Oligomers with 35S-CTPĦ. In Situ of Frozen Sections Using 35S Riboprobesģ. Selected Bibliography-In Situ Hybridization Protocolġ. In Situ PCR or PCR and In Situ Hybridization?ĭ. Hybridizations Using Synthetic Oligomersġ2. Modifications for Paraffin Sections or Cultured Cellsħ. BASIC IN SITU HYBRIDIZATION PROTOCOL (continued)Ħ. DVA Concepts in Molecular Medicine: Course Outline In situ hybridizationĭVA - Concepts In Molecular Medicine, April 8 - 13, 1996ĭepartment of Medicine, Division of Hematology/Oncology,ī.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |