Overcoming soil matrix interference for reliable environmental monitoring of dsRNA

Abstract

The extraction of high-quality double-stranded RNA (dsRNA) from soil is essential for environmental monitoring of RNA interference-based agricultural technologies and molecular ecology studies, yet it is hindered by potent nucleases and enzymatic inhibitors prevalent in soil matrices. Standard TRI Reagent®-based protocols often yield degraded dsRNA unsuitable for downstream applications. The present study systematically optimized a dsRNA extraction method for challenging, heavily clayed, and sandy soils. Initial modifications, including extended lysis times and mechanical bead beating, proved detrimental to dsRNA recovery. However, successful optimization required a multi-faceted chemical approach: incorporating β-mercaptoethanol (β-ME) to inhibit nucleases, polyvinylpyrrolidone (PVP) to adsorb inhibitors, and crucially, aluminum sulfate to effectively remove persistent PCR inhibitors. Further refinements included reducing the initial soil input and optimizing isopropanol precipitation conditions. The final optimized protocol consistently recovered approximately 80 % of spiked dsRNA (398 bp). Importantly, the extracted dsRNA was free from PCR inhibitors and suitable for sensitive downstream analysis via quantitative Real Time PCR (qRT-PCR), achieving lower limits of detection of 0.04 ng/g in clay soil and 0.004 ng/g in sandy soil. This robust and sensitive method provides a valuable tool for reliably quantifying dsRNA in complex soil environments.