IMPROVED IN-SITU HYBRIDIZATION TO HIV WITH RNA PROBES DERIVED FROM PCR PRODUCTS

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequ ences could produce more specific and intense in situ hybridization si gnals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. in situ hybridization using a pool of s even anti-sense or sense PCR-derived RNA probes (1805 nucleotides of H IV sequence, 257 nucleotides average probe length) was compared with h ybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). Th e pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves t he feasibility of synthesizing multiple, discrete RNA probes for studi es of specific mRNA expression because it does not require the subclon ing steps used to construct plasmids. PCR-derived RNA probes may provi de a viable alternative to the use of plasmid-derived RNA probes for i n situ hybridization.