October 1, 2012 (Vol. 32, No. 17)
Josh P. Roberts
Say “nucleic acid sample preparation” these days, and you could be talking about anything from a manual phenol-chloroform extraction of total RNA to a hands-free system that delivers a report of the organisms found in a sample of dirt.
Or perhaps it’s a reference to what doesn’t need to be done as, for example, when a sample can be processed directly from serum or whole blood.
A number of researchers will gather at the Knowledge Foundation Conference on “Integrating Sample Preparation: Techniques and Applications” in Baltimore later this month to address a host of sample-preparation topics.
GEN recently spoke to several of these scientists whose talks will range from discussions on novel and improved methodologies to technologies that incorporate these approaches for use in academic labs and as ready-for-market devices.
Traditionally, nucleic acid preps are designed to gather long stretches of RNA and/or DNA, with those less than 50 nucleotides considered merely uninteresting fragments. Although that view has drastically changed in the past decade or so, most protocols for extracting RNA still purposefully get rid of diminutive species like the ~22 nucleotide miRNA.
Those protocols that do specifically include small nucleic acids typically include centrifugation and/or filtration steps, notes Bee-Na Lee, Ph.D., senior applications scientist at Beckman Coulter Life Sciences.
“These methods usually do not produce consistent yield of miRNA for downstream applications, and they are not very amenable to high throughput.”
To rectify this and allow miRNA to be isolated from FFPE and cell culture samples in an automated fashion, Dr. Lee modified the binding and rebinding buffer conditions used with Beckman Coulter’s Agencourt FormaPure and RNAdvance Cell v2 kits, respectively.
These kits utilize the company’s solid-phase reversible immobilization (SPRI) technology. SPRI’s negatively charged carboxyl-coated magnetic beads would normally repel the negatively charged nucleic acids.
However an aqueous pocket is created by using a “crowding reagent” which allows the nucleic acids to move to the polar phase and reversibly bind to the beads in the presence of binding buffer. After exposure to a magnetic field, the beads that bind nucleic acids will pull to form a ring at the bottom of a well.
“You can remove all the contamination. Aspirate everything out, touching the tip to the bottom,” explains Dr. Lee. The DNA or RNA is then eluted with buffer that is “mainly just water,” preventing inhibition of downstream applications that can occur with other protocols.
For small RNA expression applications, “we normally will put the total RNA in … our yield is so high we don’t require enrichment,” she continues.
For this she credits the SPRI technology, which in addition to proprietary reagents utilizes homogenous-sized beads that are slow to aggregate or sediment, alleviating the need to frequently re-suspend. A large surface area:mass ratio allows for a high binding capacity, thus allowing the beads to rapidly respond to the magnetic field.
Taking on Sepsis
When looking for a needle in a haystack—or a single bacterium in a milliliter of blood—“the main problem becomes sample prep,” explains Sergey Dryga, Ph.D., vp of immunology at nanoMR.
Because the concentration of microorganisms responsible for sepsis is low—on the order of one colony forming unit (CFU) per mL of infected blood in bacteremia, for example—rapid tests using small volumes of blood do not have the sensitivity to deliver a statistically significant result.
Currently only those starting from a positive blood culture can do so. However, the cultures must be grown for 12–48 hours before a pathogen might even be detected, and another 12–24 hours to identify the offending organism, with the delay impacting the ability and cost to successfully treat the infection, Dr. Dryga points out.
nanoMR’s pathogen capture system (nanoMR PCS) uses antibody-coated magnetic nanoparticles to pull out pathogens from blood and deliver purified DNA in less than an hour, without the need to lyse the blood or purify the bacteria, Dr. Dryga continues.
“The method is old but nobody knows how to do it in blood, because blood is a very complicated matrix. Our innovation is that we basically developed bead chemistry, antibodies, and conditions that work in blood.”
To use the nanoMR PCS a 10 mL vacutainer of blood is delivered to a VCR tape-sized disposable cartridge containing all reagents necessary for the extraction, and the cartridge is placed in the instrument. After 50 minutes a tube is ready to be removed and used for PCR.
There is currently a list of 19 organisms that the system will recognize, including Gram-negative and Gram-positive bacteria as well as the C. albicans fungus, accounting for 96% of all bloodstream infections.
The company expects the system to be fully automated by the end of the year, and to begin trials in Europe in Q1 of 2013.
While nanoMR’s initial primary market is likely to be hospital clinical microlabs, Integrated Nano-Technologies (INT) aims to create a fully automated field laboratory using cartridges and a generic platform. Input can be a wide variety of samples: blood, tissue, insects, soil, or air filters.
“The fluidic cartridges and the devices that we’ve developed allow us to do a lot of the basic techniques you find in a laboratory,” explains INT’s president and CEO Michael Connolly, Ph.D. These include ultrasonic and chemical disruption, filtration, magnetic separation, washing and concentration of nucleic acids or proteins from a sample, small column desalting, or purification.
“And then we do PCR amplification in the cartridge, and then take that material to the detector in there.”
The company will initially produce two fully integrated units: one battery-powered, and plug-in (with battery back-up) capable of running ten tests simultaneously. Each has an integrated barcode reader and is GPS-, WiFi-, cellular-enabled, which allows them to be deployed on ships and in remote outposts.
Applications not requiring regulatory approval are expected to be available by year’s end. In one such application, the cartridge will contain a panel capable of recognizing the major mosquito-borne disease pathogens, including the alpha-, flavi-, and bunya-family viruses, dengue, and malaria.
“So you can drop the mosquitoes in there and DNA will be taken out, cleaned, amplified, and taken to the sensor, and read. The results will then be reported to you,” says Dr. Connolly.
The company will pursue three market segments. Much of their funding has come from the U.S. Department of Defense for military/security applications, and the company has a multiplexed test for biothreats including anthrax in the offing, for example.
They also plan to pursue the veterinary market and, as a longer-term goal, human diagnostics. The latter, Dr. Connolly points out, overlaps with the military market in that applications will be designed to test deployed soldiers for endemic infections.
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.