The characterization of small RNA molecules, particularly microRNAs (miRNAs), has become hugely important in the scientific community over recent years. miRNAs are ribonucleic acids made up of between 18 and 40 nucleotides, with the majority composed of around 22 nucleotides. Currently, most miRNA extraction methods co-purify miRNA and total RNA, meaning recovered samples still contain ribosomal RNA, messenger RNA, and only a low percentage of miRNA. For more accurate measurement of miRNA and higher quality miRNA sequencing data, techniques to remove unwanted ribosomal and other types of RNA are therefore highly desirable.
Beckman Coulter scientists have recently described the SPRIselect® reagent kit for the enrichment of miRNA from total RNA. This Solid Phase Reverse Immobilization (SPRI) magnetic bead method purifies nucleic acids without any centrifugation or filtration steps being required. The method involves three binding steps. First, large ribosomal RNAs are bound by the SPRI beads, with smaller fragments left in solution. The supernatant is then transferred for second binding, where intermediate RNA fragment sizes are immobilized on the beads and only the smallest RNA fragments are left in solution. The supernatant is then transferred for third binding, where the small RNAs and miRNAs are immobilized by the beads.
The SPRIselect method provides significantly more miRNA enrichment than a column method. A comparison of SPRIselect and the RNeasy® MinElute® Clean up kit showed that the MinElute column preferentially bound 53-base oligonucleotides and was less effective at binding 22-base oligonucleotides, whereas SPRIselect was more effective at binding 22-base oligonucleotides.
Using twenty human RNA samples, comparison of the two methods showed that SPRIselect achieved an average miRNA enrichment of 61.63% compared with just 51.33% using the MinElute column. SPRIselect successfully excluded fragments larger than 100 nucleotides, whereas samples recovered from the MinElute column still contained fragments of 100 to 150 nucleotides. SPRIselect also provided more flexibility in the recovery of different RNA fragments; using the three binding steps described, the supernatant can be saved and all RNA recovered during the enrichment process.
The SPRIselect reagent kit is a rapid, simple and automation friendly method that provides more effective miRNA enrichment when compared with a column method. It demonstrates a higher miRNA binding efficiency, is more efficient at separating different fragment sizes and enables the user to retain all fractions of RNA species during the enrichment process.
About Beckman Coulter
Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.
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