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.