MicroRNAs (miRNAs) are non-coding RNAs, 18-26 nucleotides long. They play an important role in the regulation of post-transcriptional gene expression by pairing with target mRNAs, causing significant modulations to cellular processes, such as cellular signaling, metabolism, cell differentiation and proliferation by adjusting protein levels. Recent studies have unveiled unique miRNA signatures in various diseases, in particular cancer, neurology, cardiology and in sepsis, which has led to propose the use of miRNAs as biomarkers. Although still far from clinical practice, (no FDA approved test using miRNA exists) this possibility has generated a strong interest and nowadays biomarker discovery is a common topic in many research projects investigating the utility of miRNA in diagnostics and prognostics.
Research trends into miRNA distribution profiling, to understand pathological and physiological mechanisms, have moved from tissues to body fluids such as plasma and serum. The surprising stability of circulating miRNAs in cell-free body fluids and their selection and packaging into secreted vesicles called exosomes, has meant that they are easier and less invasive to collect, when compared with traditional biopsies. Typically, the amount of miRNAs recovered from serum or plasma samples is low and in the picogram range. This makes those sample types very challenging to work with and the choice of RNA extraction and library preparation methods have the most significant effect on the miRNA profiles ultimately detected.
NEXTFLEX® Small RNA-Seq Kit v3 is a library prep kit solution well known for miRNA sequencing. However, when working with low inputs, such as those found in plasma and serum, it requires a gel purification step that is time consuming and could lead to loss of miRNA diversity. To address this issue, we developed the completely gel-free NEXTFLEX® Small RNA-Seq Kit v4, which has been optimized for challenging samples such as plasma and serum as well as exosomes.
In a recent study we have also presented a simplified, commercially available protocol which details exosomal RNA (exoRNA) extraction and preparation of exosomal miRNA-Seq libraries from serum and bone marrow conveniently gel-free. Using this workflow, researchers can achieve a higher number of mapped reads aligning to mature miRNA in conjunction with dimer reduction technology so that unique signatures can be detected, even with the very low miRNA inputs characteristic of exosome samples.
Additionally, the protocol is completely automatable from library prep to normalization pooling on the Sciclone® G3 NGS/NGSx and Zephyr® G3 NGS workstations.
With exceptional miRNA discovery, and a streamlined, fully automated, and completely gel-free protocol, we are confident this solution will help advance your miRNA research to the next stage.
To see data presented in a video and application notes visit this page.
For research use only. Not for use in diagnostic procedures.