Even use-as-directed assays may require some adaptation based on a host of different considerations. What type and how many samples are to be run, and from what matrix?
How precious are the samples? Is throughput as important as accuracy and reproducibility? How many parameters need to be assayed, and will it be done in monoplex or multiplex? How sensitive and selective does the assay have to be, and over what dynamic range? Will it give consistent results across lots—and can you prove it?
Scientists gathered recently at CHI’s “Biomarker World Congress” to share their insights about developing assays to measure DNA, protein, and even RNA.
There are well-established, reliable methods to visualize where specific proteins and DNA sequences lie in a tissue or in a cell: to wit, respectively, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). “Yet there’s a lack of technology to look at RNA in situ,” commented Yuling Luo, Ph.D., founder, president, and CEO of Advanced Cell Diagnostics (ACD). “That is the space that our RNAScope technology fills.”
RNA in situ hybridization (ISH) has undergone incremental improvements over the past 40 years, but not enough to endow it with the sensitivity, specificity, robustness, or simplicity to be routinely used for biomarker analysis and diagnostic applications, Dr. Luo noted. Boosting signal at the same time boosted the background, he said, yielding only limited improvement in signal:noise ratio.
ACD designed a system “that allows selective amplification of target-specific signal without amplifying nonspecific hybridization signal,” Dr. Luo explained. Two independent oligonucleotide probes simultaneously bind adjacent RNAs targets, and are recognized by a single specific “pre-amplification” molecule.
This, in turn, is bound by up to 20 amplifier molecules, each having 20 binding sites for the label probe—making a “Christmas tree-like hybridization structure”. And since a 1 kb stretch is typically targeted by 20 probe pairs, an RNA molecule can be decorated with 8,000 labels.
All the RNA resides in the cytoplasm and the assay conditions are all the same, so RNAScope can easily be multiplexed, Dr. Luo pointed out. By contrast, “in immunofluorescence analysis, because there are membrane proteins and nuclear proteins, the assay conditions are different—you have to get different antibodies in different locations to work together, which is much harder to do.”
RT-PCR, on the other hand, can assay single RNA molecules but fails to deliver tissue context. And this would be a problem when searching for loss of expression as a tumor biomarker, for example, since “grind-and-bind” methods such as RT-PCR cannot differentiate whether the expression is found in tumor or stroma.