The standard protocol of RNAscope™ 2.5 LS Reagent Kit (on the Leica Biosystems’ BOND RX Research Advanced Staining System) is designed to work for the majority of FFPE tissues.
In the study, to achieve optimal detection of RNA molecules in each tissue type, two different pretreatment conditions were compared — standard and mild — with a modification to the epitope retrieval step (Figure 2).
Standard pretreatment
BOND Epitope Retrieval Buffer 2 (ER2) at 95°C for 15 min, followed by protease digestion at 40°C for 15 min.
Mild pretreatment
ER2 at 88°C for 15 min, followed by protease digestion at 40°C for 15 min.
The optimal pretreatment condition for each tissue type was determined based on RNA signal level and the integrity of morphology. Table 4 describes optimal conditions determined in this study for tissues from rat, dog, and cynomolgus monkey. Complete data with tissue images and recommended conditions for all samples are shown in section 2. The background signal, evaluated by the bacterial dapB probe, was very low or absent in all test samples with both pretreatment conditions. To optimize conditions for species other than rat, dog, and cynomolgus monkey, it is generally recommended to start with the default pretreatment setting, which is mild for lymphoid tissues and retina and standard for other tissues.
