Optimized Method for Extracting Circulating Small RNAs from Long-Term Stored Equine Samples

Small RNAs play an important role in shaping a cell’s transcriptome profile. One miRNA may affect the expression of multiple target genes. Moreover, it has been suggested that circulating miRNA may act as hormones and be transported between cells. MiRNAs are promising biomarkers for many diseases, including cancer. MiRNA circulating in the blood are relatively easy to sample and their high stability enables implementation of simple procedures for future diagnostics. However, extracellular miRNA are only present in low concentrations and this still poses challenges regarding these procedures.

While small RNA extraction methods have been established for model organisms like human and mouse, little is known regarding optimized procedures for equine miRNA extraction. Species-specific differences in the concentration and composition of serum small RNAs have been reported.

In the presented study we optimized a method for the extraction of small RNA from equine serum. Moreover, six small RNA samples isolated with the presented protocol were then used for library preparation and successfully sequenced on an Illumina MiSeq. The sequencing data confirmed expression of some predicted equine miRNA sequences available in miRBase. The method also showed high efficiency with serum samples stored for many years.

Next, we optimized a total RNA (including small RNA) extraction method from frozen equine EDTA blood samples. In addition, we evaluated the stability of total RNA from frozen EDTA blood samples after long-term storage (> 10 years). We will present the procedure optimization steps and the most recent results at the PAG meeting.