Control slides and control probes to assess technique, sample quality and RNA quality
Success with any assay begins with good and consistent quality control practices. RNAscope® in situ hybridization is a nucleic acids based method and so it is easy to incorporate rigorous controls into every assay. ACD recommends two levels of quality control practices:
Technical quality control check— RNAscope® is an easy single-day assay, similar in workflow to immunohistochemistry (IHC) assays. The highly specific and sensitive nucleic acid hybridization this is made possible with RNAscope does require that the protocol be followed closely. Ease and familiarity with similar methods can lead one to stray from ACD’s proven protocol. To ensure success the first time and every time in specific detection of your intended target with RNAscope®, ACD strongly encourages all users to perform a technical quality control to ensure that the assay is being performed properly. This technical control is done easilyusing a cell pellet control sample tested on two slides with a low-copy housekeeping gene positive control probe and a non-specific bacterial negative control probe. When the assay is run properly, these will show strong positive control probe staining and clean negative control probe staining. We do this with every assay to verify that it is being run with correct technique.
Sample/RNA quality control check— RNAscope® Assay has universal assay conditions. Every target-specific probe uses identical hybridization conditions. However, tissue RNA quality and fixation conditions often vary. As a result, it is occasionally necessary to adjust the pretreatment conditions for optimal RNA exposure and detection by RNAscope® ISH. Empirically, we have observed that the vast majority of properly fixed tissues are suitable for RNA detection with RNAscope ISH. ACD offers guidelines on pretreatment conditions for certain tissue types. However, we encourage you to ensure that you have optimized pretreatment conditions for your tissue samples before performing your experiments. Do this by first using the positive and negative control probes on your tissues to verify that you have high positive control signal and no negative control background.If signal is low or there is any background, this is most often improved with adjustment to pretreatment times.Once you have established that sample and RNA quality are good, you can be sure that RNAscope® ISH experiments with target-specific probes will be successful.
RNAscope® In Situ Hybridization Assay Workflow
Tissue sections or cells are fixed onto slides and pretreated with RNAscope® Pretreatment Kit to unmask target RNA and permeabilize cells.
Designed with ~20 target-specific double Z probes, RNAscope® Probes hybridize to target RNA molecules.
RNAscope® Detection Reagents amplify the hybridization signals via sequential hybridization of amplifiers and label probes.
Each punctate dot signal represents a single test target RNA molecule and can be visualized with a microscope.
Single-molecule signals can be quantified on a cell-by-cell basis by manual counting or automated image analysis using HALO Software or RNAscope® SpotStudio™ Software.