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. BaseScope™® in situ hybridization assay is a nucleic acids based method in which rigorous controls can be easily incorporated into every assay. ACD recommends two levels of quality control practices:

Technical quality control check— The BaseScope™ Assay® is an easy single-day assay, similar in workflow to immunohistochemistry (IHC). The highly specific and sensitive nucleic acid hybridization, requires that the protocol be followed closely. To ensure first time and continued success in the specific detection of your intended target with BaseScope™ assay®, ACD strongly encourages all users to perform a technical quality control to ensure that the assay is being performed appropriately. This technical control is easily done using 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. With proper execution of the assay, results 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— The BaseScope™ Assay has universal assay conditions. Every target-specific probe uses identical hybridization conditions. However, variations in tissue RNA quality and fixation conditions often occur. As a result, it is necessary to adjust the pretreatment conditions for optimal RNA exposure and detection by BaseScope™ ISH. Empirically, we have observed that the vast majority of properly fixed tissues are suitable for RNA detection with BaseScope™ 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 BaseScope™ ISH experiments with target-specific probes will be successful.

RNAscope® In Situ Hybridization Assay Workflow

  • Step 1:

    Permeabilize

    Tissue sections or cells are fixed onto slides and pretreated with RNAscope® Pretreatment Kit to unmask target RNA and permeabilize cells.

  • Step 2:

    Hybridize

    Designed with ~20 target-specific double Z probes, RNAscope® Probes hybridize to target RNA molecules.

  • Step 3:

    Amplify

    RNAscope® Detection Reagents amplify the hybridization signals via sequential hybridization of amplifiers and label probes.

  • Step 4:

    Visualize

    Each punctate dot signal represents a single test target RNA molecule and can be visualized with a microscope.

  • Step 5:

    Quantify

    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.

BaseScope Negative Control Probes

ACD negative control probes ensure that there is no background staining related to your BaseScope® assay and that your tissue specimen is appropriately prepared. ACD developed a universal negative control with probes targeting DapB gene (accession # EF191515) from Bacillus subtilis strain SMY, a soil bacterium.

Alternative negative control probes can also be made-to-order at your request for your gene of choice in the sense direction (BaseScope probes are antisense) or scrambled probes. We discourage the use of sense-probes because sometimes transcription does occur on the opposite strand (and many of these events are not very well documented), in which case the results with what is intended to be a negative control to an antisense RNA may be ambiguous. Alternatively, you can apply probes from unrelated species, for instance use a zebrafish probe on human tissue.

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