MicroRNAs (miRNAs) are small ribonucleic acids with an approximate size of 22 nucleotides that is found abundantly in mammals as well as in other organisms. These non-coding RNAs are known to play an important role in many biological processes, although the precise molecular function of miRNAs in mammals is yet to be defined. More than 1,000 unique sequences of miRNA have been identified in the human genome, and it is estimated that miRNAs could target and regulate over 60% of human genes. In human cells, miRNA complementarily binds to the 3’ un-translational regions (3’UTRs) of messenger RNA and this binding plays a significant role in gene silencing and transcriptional/translational regulation in cellular responses involved in developmental processes, signal transduction, cancer and disease control.
Recently, biomedical research has placed great importance on characterizing miRNA to further understand its biological functions, and to determine how it can be used as a biomarker for cancer development and prognosis, as well as insight into how it might be used as a therapeutic target for cancer treatment. With the high demand and fast growth of miRNA research, it is necessary to develop an efficient and robust miRNA purification method to enhance downstream application assays. However, the sample preparation process for extracting miRNA from cells and tissues can be very challenging because most commercial nucleic acid extraction kits and protocols are inefficient when recovering small nucleic acids such as miRNAs. In this application note, we describe an efficient and robust miRNA extraction method using Agencourt SPRI (Solid Phase Reverse Immobilization) technology and Biomek automation.
The SPRI procedure is an easy, rapid, high yield, and automation-friendly nucleic acid purification method that does not require organic solvents, centrifugation or filtration steps. This method uses carboxyl-coated magnetic particles that reversibly bind nucleic acids in the presence of binding buffers and crowding reagents. Typically, there are three basic steps in the extraction/purification procedure. In the first step, nucleic acids are immobilized onto the magnetic SPRI beads, leaving contaminants in solution. In the second step, contaminants are removed and nucleic acids are washed, after a magnetic field is used to pull the micro-particles out of solution. Contaminants are aspirated and nucleic acids are thoroughly washed with molecular biology-grade ethanol.
In the third step, purified nucleic acids are easily eluted from the micro-particles under aqueous conditions, which provide maximum flexibility for downstream applications. This application note describes two methodologies: (1) miRNA extracted from cell culture using the Agencourt RNAdvance Cell v2 Kit and (2) miRNA extracted from FFPE.