Quest for a reliable, valid, and sensitive in situ hybridization procedure to detect viral nucleic acids in the central nervous system

Abstract

In situ hybridization (ISH) to detect and to quantitate viral nucleic acid sequences in cryopreserved central nervous system (CNS) tissue is a reliable, valid and sensitive molecular technique. On the other hand, utilization of formaldehyde fixed paraffin embedded (FFPE) tissue to improve cytomorphology requires fundamental changes in the procedure since it is necessary to cleave the elaborate protein network cross-linked by formaldehyde using elevated concentrations of proteinases in order to permit diffusion of complementary DNA probes to the targets (genomic viral nucleic acid sequences and/or viral mRNA). Adversely, this procedure hydrolyzed the proteinaceous glues generally used to fix tissue to glass slides resulting in loss of tissue sections during the ISH protocol. This report describes the application of a novel procedure utilizing a silano-organic compound to covalently bond to glass slides FFPE sections as well as cryopreserved tissue sections and cultured cells with and without virus infections. This covalent bonding procedure has permitted optimization of the ISH procedure for virus detection and quantification, especially for exploratory studies of specificity and wash stringency in relation to the Tm of the hybridized product. Progressive multifocal leucoencephalopathy (PML) caused by an opportunistic papovavirus (JC) was chosen because of the ready availability of tissue, stability of papovavirus nucleic acids, and specificity of 3H- and 35S-radiolabeled JC cloned DNA probes. Further, this laboratory is utilizing the optimized sensitive procedure to search for several virus etiologies in human diseases such as multiple sclerosis, temporal lobe epilepsy, Alzheimer's disease, schizophrenia, and Parkinson's disease, as well as normal aging.(ABSTRACT TRUNCATED AT 250 WORDS)