Diving into miRNA Detection
Signs of organ disease may be in the blood long before other phenotypic signs are evident. The kidney and liver, for example, release abundant amounts of miRNA indicating that the organ is no longer healthy, explains Martin Hegner, Ph.D., professor in the Centre for Research on Adaptive Nanostructures and Nanodevices at Trinity College Dublin.
Similarly, such miRNAs could be used to confirm a diagnosis in an emergency situation.
Dr. Hegner has been working for the past decade on ways to detect soluble macromolecules with small cantilevered array sensors. At the Knowledge Foundation conference he will describe work undertaken in collaboration with Hoffman LaRoche using these springboard-like nanomechanical sensors to specifically detect small RNA from serum using cantilever array sensors within 10–15 minutes.
“You have a sample from cells, you lyse the cells, then you sediment the debris and inject the supernatant.”
Because miRNA in blood may be present at concentrations of up to 200 million per milliliter and quite stable (as opposed to its longer RNA cousins), it should in principle be quite easy to measure, “This is not something where you’re going for single molecule detection,” he notes.
The sensors are coated with matching sequences which detect bound complementary miRNA in a couple of ways, with no labeling or modification of the target necessary, either by bending of bar, or by changing the bar’s oscillation. The technology was initially derived from scanning probe microscopy, and delivers sensitivity at the Angstrom level that can be read out using laser optics.
Any kind of small mechanical sensor will react to its environment and so it’s mandatory to include reference sensors “which are able to deconvolute any kind of background nonspecific binding from the real signal we are looking at,” explains Dr. Hegner. “We always have a minimum of two sensors. It’s a differential readout.”
The team is also collaborating with the California Institute of Technology to develop a version using integrated nanoelectronics in the springboard itself “where we don’t need an optical readout,” he says, with the aim being the creation of an entire system in a handheld device, perhaps even for use in an ambulance.
Preparing samples for Eureka Genomics’ Mass Genotyping by Sequencing Technology methodology starts with heating DNA to melt it apart and break it into smaller pieces, “which makes the hybridization of the next set of probes onto it much easier,” explains John Curry, Ph.D., senior scientist.
For each single nucleotide polymorphism (SNP) to be interrogated three barcoded probes are then added: a phosphorylated right hybridization sequence, and two left hybridization sequences, which differ by a complementary SNP and permit discrimination between the two different alleles in the genomic sequence.
The hybridized probes are then ligated and act as a template for the subsequent PCR reaction, which further adds sample specific indexes.
The assays (one sample, but hundreds of loci, per well) begin in 384-well plates and are combined and spun down into a small library “so we’re really taking a few milliliters of PCR products and reducing it down to 100 µL of library,” explains Dr. Curry. “And a portion of that library goes into the sequencer.”
This type of ligase discrimination for SNPs has been done for 20 years. Yet “whereas before people would do this assay one at a time, or 40 at a time, and resolve it on a PAGE gel, we’re resolving it on a next-gen sequencing platform,” he continues.
“So we’re able to put thousands of samples, with hundreds of loci, into a single tube, onto a single lane of an instrument, and get the information back, and decipher it, and determine the genotypes for basically 1,000 x 100 SNP-sample combinations.”
The assay never actually has to read the biological information itself “because they’re all on the probes that are designed from the biological information,” notes Dr Curry. This allows for shorter, more economical reads. In addition the barcodes can be multiplexed.
“We’re able to drive the cost down to fractions of a cent per animal SNP-combination, and do them all at once.” Eureka Genomics has already commercialized this process for agricultural and clinical applications.