Antibody Validation & Challenges
RNAscope™ Applications
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:
- Reproducibility crisis: Blame it on the antibodies. Baker M.Nature. 2015 May 21;521(7552):274-6.
- Reproducibility: Standardize antibodies used in research. Bradbury A, Plückthun A.Nature. 2015 Feb 5;518(7537):27-9
- Validating Antibodies: An Urgent Need. The Scientist. 2014 December
- Antibody validation. Bordeaux J1, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J, Anagnostou V, Rimm D.Biotechniques. 2010 Mar;48(3):197-209
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.
