Why RNA ISH application

Dual ISH and IHC

NEW! Introducing our protease-free pretreatment workflow for sequential RNA-Protein detection

 

Analysis of both RNA and protein provides invaluable information for understanding the regulation of expression. Both ISH and IHC technologies share common features that make them unique amongst the protein and RNA detection technologies.

ISH-IHC App Note

Learn more in our RNAscope ISH-IHC Application note.

Fill out the form below to download the application note:

Related Publications

Download our latest Dual ISH-IHC publication list with more than 300 publications.

Download Publication list

Related Protocol

Download our RNAscope LS Multiplex Fluorescent Assay Combined with Immunofluorescence Technical Note.

Download Technical Note

Download our RNAscope Multiplex Fluorescent Assay Combined with Immunofluorescence Technical Note

Download Technical Note

Download our RNAscope RED Assay and Immunofluorescence Technical Note

Download Technical Note

 

How RNA ISH and IHC can complement each other

To fully understand molecular and cellular mechanisms, researchers often use a variety of technologies. RNA ISH and IHC allow analysis of RNA and Protein, respectively, while also preserving the cellular relationship and tissue architecture. Researchers have found that combining or multiplexing both technologies provides unique information as described in the example below. 

RNAscope ISH validates antibodies and is a powerful alternative

IHC is a widespread technology and remains the gold standard for a broad range of application going from discovery to diagnostic and prognostic application. Nevertheless, this technology is often not without issue due to lack of manufacturing standardization and poor characterization leading to extensive validation and questionable results. The need for standardization of antibody based assays and development of antibody validation guidelines have been highlighted in many publications:

Why use RNAscope™ ISH as an alternative method to IHC?

1. Data quality

"We were very excited that RNAscope™ worked as we had had a horrible struggle with bad antibodies - We actually tested 13 different antibodies with different conditions and didn't get trustworthy results- so the RNAscope assay saved us”

Emmi Rotgers, Institute of Biomedicine/Physiology, Turku, Finland

 

- " even though we observed a significant correlation between PDL1 mRNA expression as detected by RNAscope in situ hybridization and PD-L1 protein expression as detected by IHC using two different antibodies, the signal to noise ration of RNAscope™ probe was far better that observed with IHC detection."

Increased expression of the immune modulatory molecule PD-L1 (CD274) in anaplastic meningioma. Du Z, Abedalthagafi M, Aizer AA, McHenry AR , Sun HH, Bray MA, Viramontes O, Machaidze R, Brastianos PK,Reardon DA, Dunn IF, Freeman GJ, Ligon KL, Carpenter AE, Alexander BM, Agar NY, Rodig SJ, Bradshaw EM,Santagata S. Oncotarget. 2015 Mar 10;6(7):4704-16

 

- "the COL11A1 protein levels and pattern of expression in the serial sections were consistent with COL11A1 RNA levels and pattern of expression; however, in situ hybridization provided a higher resolution signal at a cellular level."

A collagen-remodeling gene signature regulated by TGF-β signaling is associated withmetastasis and poor survival in serous ovarian cancer. Cheon DJ, Tong Y, Sim MS, Dering J, Berel D, Cui X, Lester J, Beach JA, Tighiouart M, Walts AE, Karlan BY, Orsulic S.Clin Cancer Res. 2014 Feb 1;20(3):711-23.

 

2. Speed

With just 3 weeks from sequence submission to probe delivery in your lab and reliable and reproducible performance guaranteed on positive target samples, RNAscope™ ISH becomes a very attractive option, particularly when the true costs of rigorous antibody developement and validation standards are given full consideration. Why wait for an antibody to be developed and validated, get started now with RNAscope™ ISH to accelerate your research.

 

3. Any Target

The ACD proprietary probe design algorithm allows to probe design for any gene with an unique sequence of 300 base pairs in any species including engineered animal models like knockout mouse model, xenograft models, transgenic models or humanized mouse. Researchers are no longer limited to investigating only targets with available antibodies, they routinely apply RNAscope ISH to targets with no antibodies or poor-quality antibodies.

Why use RNAscope™ ISH to validate IHC?

1. Data quality

Custom antibodies have highly variable performance. Antibody manufacturers have noted reproducibility challenges - sometimes the mouse antibody performs well initially but not so well in subsequent batches. Batch-to-batch variations, more common in polycolonal and monoclonal antibodies can be attributed to hybridoma cell-line drift, gene loss or mutations. Variability in reproducibility and specificity of antibodies have lead researchers to validate custom antibodies with RNAscope in situ hybridization.

 

2. Speed

Development of custom antibodies typically cost $20,000 and take six to nine months, yet performance is not guaranteed. ACD's customers in Biotech and Pharmaceutical companies have cited that for various reasons, they must apply IHC as the primary gene expression analysis method and thus apply RNAscope ISH as a validation method. Before spending additional time experimenting with their precious samples, they first test their custom antibodies with RNAscope ISH assays.

 

3. Unlimited targets

Every protein with a corresponding mRNA with more than 300 base unique sequences can be validated with RNAscope ISH. Submit your sequence of interest and in just 3 weeks you can validate your antibodies.

A Powerful Partnership

Immunohistochemistry (IHC) and RNA in situ Hybridization are 2 widely used technologies sharing the unique capacity to analyze a marker at the single cell level while preserving the morphological context. In many differente situations, IHC and ISH are used in conjuction to validate data or provide complementary information. 

 

X
Contact Us

Complete one of the three forms below and we will get back to you.

For Quote Requests, please provide more details in the Contact Sales form below

Advanced Cell Diagnostics

Our new headquarters office starting May 2016:

7707 Gateway Blvd.  
Newark, CA 94560
Toll Free: 1 (877) 576-3636
Phone: (510) 576-8800
Fax: (510) 576-8798

 

Bio-Techne

19 Barton Lane  
Abingdon Science Park
Abingdon
OX14 3NB
United Kingdom
Phone 2: +44 1235 529449
Fax: +44 1235 533420

 

Advanced Cell Diagnostics China

20F, Tower 3,
Raffles City Changning Office,
1193 Changning Road, Shanghai 200051

021-52293200
info.cn@bio-techne.com
Web: www.acdbio.com/cn

For general information: Info.ACD@bio-techne.com
For place an order: order.ACD@bio-techne.com
For product support: support.ACD@bio-techne.com
For career opportunities: hr.ACD@bio-techne.com