There is great interest in understanding how miRNA can be used as a biomarker for cancer development and cancer prognosis, and discovering how it might be used as a therapeutic target for cancer treatment.
With high demand for, and the fast growth of, miRNA research, an efficient and robust miRNA purification method was needed to enhance downstream assays. Most nucleic acid extraction procedures do not favor miRNA recovery, as smaller nucleic acids such as miRNA are removed during purification processes. In this article, two methodologies for recovering miRNA using the Agencourt SPRI chemistry ( Beckman Coulter Life Sciences) are described.
The solid-phase reverse immobilization (SPRI) procedure is an easy, rapid, high-yield, and automation-friendly miRNA isolation method that does not require organic solvents, centrifugation, or filtration steps. This method uses carboxyl-coated magnetic particles that reversibly bind nucleic acid in the presence of binding buffers and crowding reagents.
Typically, there are three basic steps in the purification procedure (Figure 1). In the first step, nucleic acid immobilization, SPRI beads are directly added to sample reactions. Nucleic acids are immobilized onto the SPRI beads, leaving contaminants in solution. In the second step, contaminant removal and wash, a magnetic field is used to pull the microparticles out of solution. Contaminants are aspirated, and microparticles are thoroughly washed with molecular biology-grade ethanol, yielding high-quality nucleic acids. In the third step, purified nucleic acids are easily eluted from the microparticles under aqueous conditions, which provides maximum flexibility for downstream applications.
Agencourt SPRI chemistry was designed to selectively capture larger nucleic acids; any fragments smaller than 50 nucleotides are usually removed during the binding steps. miRNAs have an approximate size of 22 nucleotides: Our initial test for miRNA isolation using the Agencourt RNAdvance Cell v2 protocol monitored the recovery of the 22 nt oligonucleotides through various binding and rebinding buffer conditions.