A rapid, cost-effective, efficient method for total RNA extraction from pure bacterial cultures and complex microbiomes and its effectiveness in deciphering functionally active microbial populations

Extraction of high-quality RNA from microbial samples, especially complex microbiomes like the gut, remains a significant challenge. The instability of RNA, coupled with the abundance of RNases and difficult-to-lyse microbial cells, often results in degraded or low-yield RNA, hampering downstream applications. To overcome this critical bottleneck, we developed a simple, rapid, cost-effective, and efficient microbial RNA extraction method, which is equally effective for pure cultures and complex microbiomes. The method utilises low pH (4.7) and high temperature to lyse the cells, followed by rapid cooling to induce misfolded renaturation of genomic DNA and proteins. During the phase separation step, RNA remains in the aqueous phase and is subsequently precipitated using chilled isopropanol. The RNA suspended in double-distilled water (pH 6.5) remained stable for six months at −20 °C. Further, we compared amplicon microbiome profiles generated from metagenomic DNA and cDNA (synthesised from extracted meta-RNA) derived from the broiler gut, taken as a model system. Our findings reveal that the cDNA-based amplicon microbiome composition more accurately deciphers the metabolically active population in gut environments that aligns with culture-based enumeration of viable microbes. This proof-of-concept study underscores the importance of meta-RNA approaches for functional microbiome analysis and provides a reliable RNA isolation method that can be broadly adopted for studies aiming to resolve active microbial community structure.