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.