Dr. Boye Schnack Nielsen provides tips for performing successful in situ hybridization tests and provides insights into the evolution of RNA detection methods over time
Dr. Boye Schnack Nielsen is the Business and Research Director of the Molecular Histology Department at Bioneer AS
Diagnostic tools in histopathology have traditionally focused on studying DNA and chromosomal abnormalities and measuring protein levels. Polymerase chain reaction (PCR) and microarrays, which use the stability of DNA and protein molecules, have become established gold standards for investigating molecular profiles in various diseases. However, molecular scientists have long recognized that if they skip the rich context that RNA analysis provides on variations in gene expression patterns, they will lose clinically relevant information.
Fill in the gaps
Eliminating this information gap is a key concern of molecular histologists such as Dr. Boye Schnack Nielsen, Business and Research Manager for Molecular Histology at Bioneer A / S. “RNAs are interesting groups of molecules, including transfer RNAs (tRNA) and ribosomal RNAs (rRNA), which are better known, along with the long non- coding RNAs (lncRNA), microRNAs (miRNA) and circular RNAs (circRNA). that have grown in importance in recent times. Traditionally, RNAs were not viewed as diagnostic tools, as RNAs can easily be lost due to degradation, ”says Nielsen. “In addition, RNA detection methods were often complicated and required the presence of qualified laboratory technicians.”
At Bioneer, a technology service provider approved by the Danish Ministry of Science and Technology, Nielsen and his team help companies overcome such challenges. Current projects include the development and optimization of RNA detection methods such as RNA in situ hybridization tests and their combination with immunohistochemical techniques. As a contract research organization, Bioneer works with its customers in biotechnology to carry out translation projects across the entire life cycle in disease biology. Such projects typically include answering questions in oncology, CNS and stem cell research, and developing and manufacturing drug candidates for preclinical studies.
The secret of success: control tests
“The key to successful RNA work is to always run control tests,” says Nielsen. “When an experiment fails, it can be difficult to narrow down the culprit to incorrectly processed samples, inadequate experimental conditions, and RNA probes with poor detection quality. Even the two types of tissue used for RNA work, frozen and paraffin-embedded tissue, can produce good results if processed properly, ”he continues. “If your assay doesn’t work as expected, you need to test various experimental conditions. To do this, rely on your control tests to fix the problem. I use a combination of internal positive control tissue to test my sample quality and control probes to test the performance of my probes. “
In the past ten years, the field of RNA diagnostics has developed rapidly and the range of applications for RNA detection techniques has expanded, says Nielsen. “Companies like Advanced Cell Diagnostics (ACD) have developed innovative methods for detecting RNA for use in histology,” explains Nielsen. “To ensure easy implementation, these RNA assays are adapted for automated use on the Leica and Leica [Roche] Ventana instruments commonly used in pathology laboratories. These assays focus on providing sensitivity and specificity and have also become more reproducible over the years. ”
One of the most popular methods for miRNA staining in clinical specimens has been LNA probe technology since its introduction in 20101. LNA technology uses nucleic acid analogs called blocked nucleic acids and uses both chromogenic and fluorescent methods to detect a range of RNA and DNA molecules. While LNA has significantly higher sensitivity and target specificity compared to DNA and RNA probes used in the past, reproducibility can still be difficult for some of the shorter RNA molecules.
A streamlined, reliable assay
To address this lack of LNA technology, the ACD assay developers created the miRNAscope ™, an in situ hybridization assay. The miRNAscope helps visualize antisense oligonucleotides (ASOs), miRNAs, small interfering RNAs (siRNAs), and other short nucleic acid targets in both intact tissues and cultured cells.
As one of the first pioneers of LNA technology, Nielsen knew that the miRNAscope first had to be a real improvement on LNA technology in order to replicate the results of LNA technology. To assess the miRNAscope, his team used targets that are typically well absorbed via LNA, such as U6, a small nuclear RNA, and microRNA-126, an endothelial-specific microRNA. And the miRNAscope passed the test with flying colors, explains Nielsen: “We know what to look for in the staining patterns: With the miRNAscope we see a clear localization of microRNA-126 in the blood vessels. In addition, we see a better output with the miRNAscope for another microRNA that was not reproducibly detected with the LNA technology. This is not entirely unexpected given the tremendous optimization efforts that have been made to make the miRNAscope more sensitive, ”he says.
Nielsen is pleased that the miRNAscope offers companies like Bioneer and their customers an alternative for the detection of microRNAs that is not only done via LNA. In the future, he predicts that miRNAscope will be a significant improvement on LNA assays, allowing better staining of a wider range of microRNAs and improving assay reproducibility. “We really appreciate the innovative people at companies like ACD, who not only focus on developing probes and improving detection technology, but are also interested in refining novel tissue processing steps,” says Nielsen. As Nielsen looks forward to using the automated version of the miRNAscope kit next, he concludes that he is confident that the full potential of RNA detection in the field of disease biology will soon be realized.
1. Jørgensen, S., Baker, A., Møller, S. & Nielsen, BS Robust one day on site Hybridization protocol for the detection of microRNAs in paraffin samples with LNA probes. Methods, 2010.
Do you use Advanced cell diagnosis Products in your laboratory? Write a review for your chance to win a $ 400 Amazon Gift Card >>