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 in which rigorous controls can be easily incorporated into every assay. ACD recommends two levels of quality control practice:

Technical assay control check— The RNAscope® Assay is an easy single-day assay, similar in workflow to the immunohistochemistry (IHC) assay. The highly specific and sensitive nucleic acid hybridization is made possible with RNAscope® technology, which requires close attention to the protocol. To ensure first-time success with specific detection of your intended target using the RNAscope® assay, ACD strongly encourages all users to perform a technical assay control to ensure that the assay is being performed appropriately. This technical control is easily performed 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. When the assay is run properly, this technical control 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 the correct technique.

Sample/RNA quality control check— The 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 provides guidelines on pretreatment conditions for certain tissue types. However, we encourage you to optimize the specific 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 the RNAscope® 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.

RNAscope Negative Control Probes

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

Alternative negative control probes can be made-to-order at your request for your gene of choice in the sense direction (RNAscope probes are antisense) or scrambled probes. We discourage the use of sense probes because occasionally 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.

Which RNAscope® Positive Control Probe is right for me?

Positive Control Probe Gene

Expression Level (copies per cell)*

Recommendations

Use with high expression targets

UBC (ubiquitin C)

Medium / High (>20)

UBC is expressed at a medium high level and should be paired with high expressing targets. Applying UBC along side with a low expressing target is not recommended as it can lead to giving false negative results. Because the RNAscope assay is so sensitive, it may be possible to detect UBC RNA even with substantial degradation and under non-optimal assay conditions.

Most flexible option

PPIB (Cyclophilin B)

Medium (10-30)

PPIB is our recommendation for use as a positive control for most tissues. It is expressed at a sufficiently low level so as to provide a rigorous control for sample quality and technical performance. In the vast majority of studies, if PPIB is positive, then any target probe will detect your target RNA. PPIB is also often used as a reference in RT-PCR.

Use with low expression targets

Polr2A (DNA-directed RNA polymerase II subunit RPB1)

Low (3-15)

POLR2A is an additional low copy, rigorous positive control; for very low expressing targets. It can be an alternative to PPIB for proliferating tissues, like tumors, and also for some non-tumor tissues (eg retinal tissue and lymphoid tissues).

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