Probe Design

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.

This depends on the sequence homology:  >95% homology is needed across species to create such probes. Please contact your local ACD account executive for information specific to your gene. If you are a new customer and would like to get started please use contact us link on our website (http://www.acdbio.com/about/contact). ACD will get back to you with the requested information

ACD probe design algorithm is validated in silico to select oligos with compatible melting temperatures for optimal hybridization at RNAscope assay conditions and minimal cross-hybridization to off-target sequences. There is a verification procedure following each major step during probe design to guarantee the accuracy. Please refer to the RNAscope technique paper for details: http://www.ncbi.nlm.nih.gov/pubmed/22166544.

Yes. The same channel 1 (C1) Ready-to-Use probe can be used in each manual assay format – single-plex for chromogenic, 2-plex chromogenic and fluorescence multiplex. 

With the duplex assay, you will need to use channel 1 (C1) and channel 2 (C2). 

With the multiplex assay (for example: 4-plex), you will need to use channel 1 (C1), channel 2 (C2), channel 3 (C3) and channel 4 (C4) probes. 

ACD provides the 5’ and 3’ nucleotide positions of the target probe region and the number of probe pairs generated to that region. This information is available as you review your probe information on the ACD website. The exact probe pair location and sequences are considered ACD’s proprietary information.

RNAscope is designed to detect any mRNA or ncRNA greater than 300 bases. A standard RNAscope probe design includes 20 ZZ pairs.

Basescope is designed to detect short target sequences that are 50 to 300 bases. The Basescope probe is designed in 1-3 ZZ probe pairs

miRNAscope detects RNAs that are between 17 to 50 bases.