Methodological considerations for aqueous environmental RNA collection, preservation, and extraction

Area of Science:

• Environmental science
• Molecular biology
• Aquatic ecology

Background:

• Environmental RNA (eRNA) analysis promises superior physiological and distribution data compared to environmental DNA (eDNA).
• The physicochemical instability of eRNA necessitates technological advancements for reliable detection and analysis.
• Zebrafish (Danio rerio) were used to validate eRNA methodologies in aquatic samples.

Purpose of the Study:

• To optimize methods for capturing, preserving, and extracting eRNA from water samples.
• To enhance the efficiency and reliability of eRNA analysis for environmental monitoring.
• To improve the accessibility of eRNA data for assessing aquatic ecosystem health.

Main Methods:

• Optimized lysis buffer volume for eRNA extraction, increasing target concentration.
• Compared GF/F and GF/A filters for eRNA capture efficiency based on filtration volume and time.
• Evaluated the efficacy of RNA stabilization reagent (RNAlater) for eRNA preservation at different temperatures.

Main Results:

• Increased lysis buffer volume significantly boosted target eRNA concentration (over sixfold).
• GF/A filters showed potential for capturing more eRNA particles due to higher filtration capacity.
• RNAlater ensured stable eRNA preservation on filters for at least 6 days at -20°C and 4°C.

Conclusions:

• Optimized extraction and capture methods increase eRNA yield and availability from field samples.
• RNAlater enables convenient eRNA sample preservation without requiring ultra-low temperatures.
• These advancements will refine eRNA analysis for biological and physiological monitoring in aquatic environments.