Faqs

ACD always recommends running 3 slides minimum per sample: your target marker panel, a positive control, and a negative control probe.  The positive control will help determine whether the quality of RNA in the tissue specimen is sufficient for detecting your RNA target. The bacterial dapB negative control will help determine whether the tissue specimen is appropriately prepared for RNAscope and BaseScope assays.

The RNAscope Multiplex Fluorescent v2 Assay and HiPlex v2 Assay can be imaged using either an epi-fluorescent or confocal microscope with the appropriate filters for assigned fluorophores. For specific excitation wavelength of each probe channel please refer to the appropriate user manual for your assay of interest.

RNAscope signal is detected as punctate dots. Clusters can result from overlapping signals from multiple mRNA molecules that are in close proximity to each other.

There may be variation in dot intensity/size which is because of the differences in the number of ZZ probes bound to a target molecule. However, since each dot represents a single transcript, the number of dots is critical and not the intensity/size of the dot(s).

RNAscope signal is shown as punctate dots. Each dot represents a single copy of an mRNA molecule.

ACD recommends maintaining RNAse-free environment while handling and sectioning tissues prior to fixation. Once the samples have been properly fixed, further RNA degradation is not expected to occur.

RNAscope, BaseScope and miRNAscope assays can be used with FFPE tissue, fresh-frozen tissue, fixed-frozen tissue, and cultured cells. ACD has user manuals and technical notes and recommendations for preparing each of these sample types. Note that cultured cell samples are not compatible with RNAscope HiPlex v2 assay.

Our probe stability is tested for up to 2 years from the date of manufacturing when they are stored as recommended at 4°C.

The C1, T1 and S1 designation is the amplification channel the probe was designed to be detected in. The letter is used to designate the assay it can be used with. Any probe with a C listed is used with our RNAscope and Basescope assays; any probes with the T designation is used with our HiPlex assay. Any probe that has the S1 in the probe names is designed for our miRNAscope assay.

In the fluorescent assay, the amplification channel number (for example: C2, C3, C4 or T1, T2, T3, etc.) allows you to multiplex your targets to be detected using various fluorophores.

Probes are designed with oligo pairs. Each oligo has two hybridizing regions, and ACD refers to these oligos as ZZ pairs. The “bottom” of the Z oligo has 18 to 25-base region that is complementary to the target RNA. This sequence is selected for target specific hybridization and uniform hybridization properties. So, each ZZ oligo pair hybridizes to 36 to 50 bases of target RNA and a typical RNAscope probe consists of 20 ZZ pairs spanning about 1000 bases of unique sequence. Redundancy and robustness are built into our design strategy resulting in high specificity.