H. Benjamin Larman  is an assistant professor within the Immunology Division of the Department of Pathology. He spoke with Fundamentals about a new technique for analyzing the RNA content of preserved human tissues. Messenger RNA molecules reflect gene expression—that is, whether and how much particular genes are being used. This can be important for determining which tumors are the most dangerous and how they interact with—and in some cases evade—the immune system. In addition, detecting RNA from microbes with this method can be used to diagnose infections, as demonstrated in a recent publication.

What is your overall research focus?

Larman: My lab uses recent advances in DNA synthesis and sequencing technology to create new molecular tests to analyze human samples. The challenge we face in studying human samples is the large amount of natural variation due to our different genetic backgrounds and environmental exposures. This means we often need to study large numbers of individuals— sometimes up to hundreds or thousands—to find significant disease associations. This makes minimizing measurement costs a top priority for us.

What led you to create this new method?

Larman: There are many research and clinical scenarios in which analyzing RNA in preserved tissues is important. When surgeons collect tissues, such as tumors, the pathologist typically fixes the sample in formalin (essentially embalming it) and embeds it in paraffin wax for microscopic analysis. However, this process irreversibly damages the genetic materials in the cells and makes it particularly challenging to measure gene expression. Existing techniques are not only unreliable for these types of samples, but also too expensive to be used for hundreds or thousands of samples. Our new method is very inexpensive when used on large numbers of samples, and it circumvents several important limitations of alternative techniques. 

What makes this method different?

Larman: Rather than extracting the RNA from the tissue and using reverse transcription to convert RNA sequences into DNA sequences, our new method, ligation in situ hybridization (LISH), uses specific probes made of DNA linked to RNA and a ligase enzyme that joins them together only when bound to their matching RNA sequences. Because this approach is more effective at measuring damaged RNA, we’re able to get more accurate results. The joined probes, which are formed inside the tissue, can then be easily recovered and amplified for analysis via high-throughput DNA sequencing. 

There are two main benefits of being able to amplify the joined probes. First, we can incorporate sample-specific “DNA barcodes,” which allows the simultaneous analysis of hundreds of individual samples. This can bring the analysis cost down to around $10 per tissue sample. Second, amplification allows us to analyze exceedingly small tissue fragments, even down to just a couple of isolated cells.

What are some other ways you expect this technique will be used?

Larman: LISH is applicable to the study of any localized disease process in any tissue type. It could be used to diagnose infections or to study neurodegenerative diseases that affect only certain cells in the brain for example—really, anything where it's important to measure the expression of lots of genes in a small, defined area of tissue.

Ultimately, we’re hoping to pair LISH with complementary technologies that we work on, in order to more comprehensively characterize human immune responses. One such example is phage immunoprecipitation sequencing (PhIP-Seq), which we use to broadly characterize the binding properties of an individual’s antibodies.

It's an amazing time to be doing this type of work because of the tools available now, particularly high-throughput DNA synthesis and next-generation DNA sequencing. We're able to do things routinely and inexpensively today that even 10 years ago people could have only dreamed about. 

 

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This work was made possible in part by support from the Johns Hopkins Medicine Discovery Fund; Jerome L. Greene Foundation; NCI IMAT Program R21 Award [CA202875]; NIAID U24 Award [AI118633]; Prostate Cancer Foundation Young Investigator Award; and a Catherine and Constantinos J. Limas Research Award.