Digoxigenin as an alternative probe labeling for in situ hybridization.
In situ hybridization (ISH) techniques have proven to be invaluable tools for diagnostic molecular laboratories in the detection of gene expression and viral infection in cell and tissue samples. Radioactively labeled probes are still widely used for ISH because of their high sensitivity in detection. Among the non-isotopic systems only biotinylated probes for viral DNA detection found broader acceptance. Recently we introduced the digoxigenin probe labeling and detection system for ISH in our diagnostic molecular laboratory. Our experiences with digoxigenin-labeled probes are summarized in this report in the form of technical recommendations for probe choice, labeling, quantification, hybridization and detection. Several ISH protocols using RNA, DNA and oligonucleotide probes for the detection of mRNA and viral DNA are reported. Advantages, disadvantages and pitfalls of the digoxigenin system as well as comparisons with radiolabeled and other non-isotopic systems are discussed. Digoxigenin-labeled probes provide an attractive alternative to radiolabeled probes and are superior to other nonradioactive probes for ISH. Probe labeling, quantification, hybridization and detection can be performed with low biohazard risk. Working efforts and costs applying digoxigenin-labeled probes or radiolabeled probes for ISH are similar. Digoxigenin-labeled probes are stable for several months, provide an equal sensitivity in detection and a higher degree of cellular resolution than radiolabeled probes. The turnaround time of procedures is short and results of diagnostic ISH can be obtained much quicker than using radiolabeled probes.
Komminoth
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Department of Pathology, Tufts University School of Medicine, New England Medical Center Hospital, Boston, Massachusetts 02111.
D004076:Digoxigenin; D004355:Drug Stability; D005069:Evaluation Studies as Topic; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods* / Q000706:statistics & numerical data; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D010280:Parathyroid Glands / Q000378:metabolism; D010281:Parathyroid Hormone / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D010281:Parathyroid Hormone; D012333:RNA, Messenger; D004076:Digoxigenin