Preserving RNA is made difficult due to presence of RNase enzyme. This enzyme is found on glassware, in reagents and on operators and their clothing. RNase quickly destroys any RNA in the cell or the RNA probe itself so users must ensure they use sterile techniques, gloves, and solutions to prevent RNase contamination of the probe or tissue RNA.
RNA probes should be 250–1,500 bases in length; probes of approximately 800 bases long exhibit the highest sensitivity and specificity. Transcription templates should allow for transcription of both probe (antisense strand) and negative control (sense strand) RNAs. Clone into a vector with opposable promoters to achieve this. Circular templates must be linearized before making a probe, though PCR templates can also be used.
This protocol describes the use of DIG-labeled single-stranded RNA probes to detect expression of the gene of interest in paraffin-embedded sections.