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RNAscope® 2.5 LS Duplex Assay

The RNAscope® 2.5 LS Duplex Assay is designed as an automated assay on the Leica Biosystems' BOND RX Research Advanced Staining System. The RNAscope® 2.5 LS Duplex Assay, is an RNA in situ hybridization assay based on ACD’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in a multitude of sample types mounted on slides. RNAscope® Assays do not require the RNA-free environment used for traditional ISH.

RNAscope® 2.5 LS Duplex Kit is designed for simultaneous in situ detection of two RNA species. 

Common applications include co-localization studies to map co-expression of two targets within the same cellular context (e.g. secreted ligand and its receptor) or to profile gene expression in a specific cell type expressing a known marker (e.g. a specific stem cell marker). To distinguish between the two targets, individual probes are used in probe channels 1 (C1) and Channel 2 ( C2). Channel 1 (C1)  is detected using HRP based diaminobenzidine (DAB) reaction resulting in a dark brown signal and Channel 2 (C2)  is detected using an AP Fast Red based reaction resulting in a red color. Due to the brightness of the red chromogen, it is advised that the lower expressed target be ordered in the C2 channel and stronger expressed target be ordered in the C1 channel.The stained slides are visualized with a bright-field microscope. 

These new RNAscope® 2.5 LS Duplex reagents require the new RNAscope® 2.5 LS Probes for optimal performance. Probe designs are identical to previous version, however the RNAscope® 2.5 LS probes are manufactured for optimal performance with RNAscope® 2.5 LS reagent chemistry.

Sample Images
Detection of Grin2C (dark brown) and Sst (red) mRNA in mouse brain tissue with RNAscope<sup>®</sup> 2.5 LS Duplex Assay (RNA ISH)

Detection of Grin2C (dark brown) and Sst (red) mRNA in mouse brain tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

Detection of Grin2C (dark brown) and Pvalb (red) mRNA in mouse brain tissue with RNAscope<sup>®</sup> 2.5 LS Duplex Assay (RNA ISH)

Detection of Grin2C (dark brown) and Pvalb (red) mRNA in mouse brain tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

Detection of Grin2C (dark brown) and Pvalb (red) mRNA in mouse brain tissue with RNAscope<sup>®</sup> 2.5 LS Duplex Assay (RNA ISH)

Detection of Grin2C (dark brown) and Pvalb (red) mRNA in mouse brain tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

Detection of Ppib (dark brown) and Pvalb (red) mRNA in mouse brain FFPE tissue with RNAscope<sup>®</sup> 2.5 LS Duplex Assay (RNA ISH)

Detection of Ppib (dark brown) and Pvalb (red) mRNA in mouse brain FFPE tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

Detection of HPV HR18 (dark brown) and P16 (red) mRNA in human head and neck cancer tissue with RNAscope<sup>®</sup> 2.5 LS Duplex Assay (RNA ISH)

Detection of HPV HR18 (dark brown) and P16 (red) mRNA in human head and neck cancer tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

Detection of NRG1 (dark brown) and ERBB3 (red) mRNA in human breast tissue with RNAscope<sup>®</sup> 2.5 LS Duplex Assay (RNA ISH)

Detection of NRG1 (dark brown) and ERBB3 (red) mRNA in human breast tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

Detection of NRG1 (dark brown) and ERBB3 (red) mRNA in human breast tissue with RNAscope® 2.5 LS Duplex Assay (RNA ISH)

                            

 

The RNAscope® 2.5  LS Automated Assay requires the following from Leica Biosystems (LeicaBiosystems.com):

 

Component

Cat. No.

BOND Open Containers 30 mL

Op309700

BOND Universal Covertiles 100 pack

S21.2001

BOND Epitope Retrieval Solution 1-1L (RTU)

AR9961

BOND Epitope Retrieval Solution 2-1L (RTU)

AR9640

BOND Dewax Solution – 1L (RTU)

AR9222

BOND Wash Solution 10X Concentrate – 1L

AR9590

BOND Polymer Refine Detection (DAB) and Hematoxylin*

DS9800

BOND Polymer Refine Red Detection*

  DS9390

BOND  DAB Enhancer 30ml

  AR9432

BOND Aspirating Probe Cleaning System

CS9100

BOND Mixing Stations

S21.1971

 

RNAscope® 2.5 LS Duplex Reagent Kit Workflow Requires the following from ACD:

  • RNAscope® 2.5 LS Target Probes (Catalog or Made-to-Order LS Probes)
    300038 RNAscope® 2.5 LS Probe (RTU)
    300038-C2 RNAscope® 2.5 LS Probe- C2 (50x)
  • RNAscope® 2.5 LS Control Probes choose from Pooled Control Probes for both C1 and C2 channels or Control probes in C2 only:
    • ​​​Pooled Control Probes

320768 RNAscope® 2.5 LS Duplex Control Probes (PPIB-C1, Polr2A-C2)- Mm

320748 RNAscope® 2.5 LS Duplex Control Probes (PPIB-C1, Polr2A-C2)- Hu

320758 RNAscope® 2.5 LS Duplex Negative Control Probe (DapB-C1, DapB-C2)

  • Control Probes for C2 only (to be used with C1 RTU Target probes)

313908-C2 RNAscope® 2.5 LS Positive Control Probe - Hs-PPIB-C2 (50X)

312038-C2 RNAscope® 2.5 LS Negative Control Probe - DapB-C2 (50x)

  • RNAscope® Control Slides (optional)
    • Human Cell Pellet Cat. No. 310045, or Mouse 3T3 Cell Pellet Cat. No. 310023
  • RNAscope® 2.5 LS Duplex Reagent Kit
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