Innovative Uses of RNAscope

Read about reseachers who take innovative approaches with RNAscope® protocols to advance their research. If you would like to try one of these customer-demonstrated applications, please note that they are non-standard applications and therefore, a careful read through of the publications/materials is highly recommended. Our technical support team is here and will make best efforts to support you.

Customer demonstrated application:
SIV/HIV RNA & DNA detection
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ACD congratulates the team of scientists, led by Jacob D. Estes on their publication Defining HIV and SIV Reservoirs in Lymphoid Tissues DOI 10.20411/pai.v1i1.100.

The authors aimed to track and discriminate HIV-1/SIV viral reservoirs within tissue compartments and thus applied a 'specific and sensitive next-generation in situ hybridization approach.' The authors demonstrated:

1) that an optimized next-generation ISH platform RNAscope Assay for the rapid detection of vRNA (with results obtained within 1 day) has sufficient sensitivity to reliably detect single virions in B cell follicles (BCF) in FFPE tissue sections,

2) that an approach for the detection of vDNA in situ (referred to as DNAscope) reliably and readily detects vDNA+ cells, and

3) that they have developed an in situ method to simultaneously visualize vRNA and vDNA in the same tissue section and thereby identify transcriptionally latent infections (vDNA+/vRNA- cells) in lymphoid tissues.

The team of researchers then applied “these new, highly sensitive in situ hybridization approaches" to lymphoid tissues samples from macaques prior to and during combination anti-retroviral therapy (cART) and documented the importance of BCFs in active, latent and persistent infections during treatment. Authors concluded that “these data underscore the utility of new and sensitive ISH tools that can provide additional insight into viral persistence, reservoir establishment, tissue compartmentalization and reservoir phenotype in the local in vivo tissue environment.”

Read the Publication

Contact ACD to get started

Visualization of viral DNA as described above is a non-standard, modified RNAscope® protocol.  Please familiarize yourself with the methodology described. The ACD technical support team will provide the best support for you.  Please note, as yet, there are no experimental data demonstrating the extension of this protocol to other species, other probes or other tissue types not described in the publication.  

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What to order from ACD

Your account executive will ensure completeness of your order.  Standard accessories needed for any of the three protocols below include:

1. ACD HybEZ Hydridization System with ACD EZBatch Cat. No. 321461 110V or Cat. No. 321462 220V

2. ImmEdge Hydrophobic Barrier Pen Cat. No. 310018

3. Control probes:  Negative control probe and species-specific positive control probes are also required 

 

To detect and visualize HIV-1 AND SIV viral RNA in situ, the authors used standard RNAscope Assay Protocol and anti-sense probes outlined in Supplemental Table 2. Additionally the following materials from ACD are required:

1.  RNAscope 2.5 HD Red Kit: Cat. No. 322350 

2. EcoMount Mounting Medium Cat. No. 320409

 

To detect and visualize HIV-1 AND SIV viral DNA in situ, the authors used modified RNAscope Assay Protocol and sense probes outlined in Supplemental Table 2. Additionally the following materials from ACD are required:

1.RNAscope 2.5 HD Brown Kit Cat. No. 322300

 

To detect and visualize both viral RNA and viral DNA in situ, in a single assay the authors used modified RNAscope Assay Protocol both sense and anti-sense probes outlined in Supplemental Table 3. The following materials from ACD are also required:

1. RNAscope 2.5 HD Duplex Kit Cat. No. 322430

2. VectaMount Permanent Mounting Medium Cat. No.321584

 

Customer demonstrated application:
WISH Whole-Mount in situ Hybridization
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Our ACD team are excited that Theresa Gross-Thebing, Azadeh Paksa and Erez Raz at Münster University, Germany have driven the RNAscope® technology into a new arena - establishing, optimizing and applying RNAscope® Assay to whole mount embryonic zebrafish samples. Although RNAscope® assay may be applied to any gene and any species, appropriate sample preparation is essential.

Contact the ACD support team at support@acdbio.com for detailed support for the of RNAscope® Assay.

Theresa Gross-Thebing and Azadeh Paksa have generously volunteered to answer questions specific to the preparation of whole-mount embryonic samples.  Email them using RNAscope4zebrafish@acdbio.com but please note, the response time may take up to 1 week.

A Tips & Tricks and FAQ section surrounding WISH is under development, please check back with us for updates.

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  WHAT TO ORDER TO GET STARTED?

  1. RNAscope® positive control probe:  use zebrafish-specific housekeeping gene for positive control. The Erez Raz Lab used zebrafish myoD gene in channel 2,  CAT # 402461-C2  Dr-myod1-C2.  Alternatively, we also offer CAT# 427301-C2 Dr-gsc-C2 and CAT# 428601 Dr-odc1 (channel 1)
  2. RNAscope® negative control probe: CAT # 310043 Negative Control Probe-DapB
  3. RNAscope® Target Probes:   ACD currently offers 33 zebrafish-specific probes (www.acdbio.com/probesearch).  New or custom target probes can be designed and manufactured within 2 weeks. 
  4. RNAscope® Assay Reagent Kit:  The Erez Raz Lab used CAT # 320850 RNAscope® Multiplex Fluorescent Reagent Kit, comprise of  three separate components that are also available separately: 
  • CAT # 310091 Wash Buffers
  • CAT # 320842 RNAscope® Pretreatment Kit 
  • CAT # 320851 RNAscope® Multiplex Fluorescent Detection Kit

A multispectral microscopy imaging system is required for visualization. You will be able to investigate up to 4 genes simultaneously with this assay.   Additionally, ACD offers three chromogenic assays; however those assays have not been applied to whole-mount zebrafish embryonic samples.

Customer demonstrated application:
Researchers Successfully Apply RNAscope ISH to 25-year-old FFPE Samples
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Innovations in the Spotlight:

Erasmus MC Researchers Successfully Applied RNAscope® ISH to 25 year old FFPE Samples

 

You may be surprised to know that your archived FFPE samples may not be too old for RNAscope® in situ hybridization (ISH)! Our R&D team routinely detects RNA transcripts in three year old FFPE tissue specimens and some of our customers have applied RNAscope to much older tissues.  Read below, data from one of our customers where RNAscope ISH was successfully used in tissue specimens older than 25 years.

Success of RNAscope® with older FFPE samples depends on many factors including sample fixation, quality and storage. ACD cannot guarantee results for samples outside of our protocol recommendations. Please read FAQs for RNAscope® assay on archived FFPE samples section below for more information.

ACD Customers at Erasmus MC Push the Limits within RNAscope® FFFPE Protocol

Researchers Marije Hoogland and Esther Verhoef (Department of Pathology, Erasmus MC Rotterdam, the Netherlands) have conducted a retrospective study and successfully performed RNAscope® in situ hybridization on samples containing human metastases of prostate cancer in a lymph node. The samples were collected in 1987, 1988 and 1989 and analyzed in 2014 with RNAscope® probe targeting UBC gene. At 25 to 27 years old, these are the oldest samples we know of that have been used with RNAscope® in situ hybridization. The team followed ACD protocols and user manuals.

FAQs for RNAscope® Assay on archived FFPE samples

How can I ensure that my FFPE tissue specimens will work with RNAscope® FFPE Assay?

We have conducted extensive assay development to make sure that the RNAscope® FFPE assay will work with a wide range of tissue types.

  • If your tissue specimens are processed according to the Sample Preparation Guide in the User Manual and they are prepared within the last 3 years, you should expect successful staining for positive control probe using our assay procedure.
  • If your specimens are prepared differently from our recommended procedure, RNAscope® can still be used but may require assay optimization for some procedural steps. Please refer to our getting started page for protocol and sample guidelines and optimization.
  • Ensure that the assay is run according to RNAscope® protocol.

What are the recommended RNAscope® sample storage and pretreatment conditions for FFPE samples?

ACD sample storage requirements can be found in our RNAscope® Sample Preparation and Pretreatment Guide for FFPE Tissue, PART 1. The recommended pretreatment conditions for FFPE samples are based on tissue that has been fixed for 16-32 hrs at room temperature in 10% NBF.

What should I do if I don’t know whether the FFPE tissue specimen is appropriately fixed and processed?

Determine your samples' RNA quality following optimization recommendations on our getting started page (www.acdbio.com/go).

Figure 1: Punctate red dots show expression of UBC gene in Human prostate cancer samples from lymph node. RNAscope Chromogenic Red assay applied to FFPE  sample from 1989 , 400x magnification.

Figure 2: Expression of UBC gene (shown as punctate red dots) in human prostate cancer samples from lymph node. RNAscope Chromogenic Red Assay applied to FFPE samples from 1989, 400x magnification.

Figure 3: Expression of UBC gene (shown as punctate red dots) in human prostate cancer samples from lymph node. RNAscope Chromogenic Red Assay applied to FFPE samples from 1987, 400x magnification.

Figure 4: Expression of UBC gene (shown as punctate red dots) in human prostate cancer samples from lymph node. RNAscope Chromogenic Red assay applied to FFPE samples from 1989, 400x magnification.

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