A trizol-based method for extracting Listeria monocytogenes RNA from raw and pasteurized milk

Extracting bacterial RNA from milk is technically difficult because fats, proteins, cells, calcium, and natural RNases interfere with bacterial lysis and phase separation. Standard RNA extraction protocols often use commercial silica-column kits, but these require large sample amounts or involve complex multi-step procedures. Here, we introduce and validate a simple, cost-effective, kit-free method to isolate bacterial RNA directly from raw and pasteurized cow's milk using a modified TRIzol-based approach. This method reliably produces intact RNA, as evidenced by clear 23S and 16S rRNA bands, A260/280 ratios between 1.94 and 2.0, and concentrations ranging from 110 to 556 ng/μL. The obtained RNA can be used in various tests, including PCR, RT-qPCR, and activation of synthetic RNA biosensors (Toehold), without interference from milk components. To our knowledge, this is the first small-volume, kit-free TRIzol method capable of recovering functional bacterial RNA directly from raw cow's milk, providing an accessible tool for RNA amplification, detection, and diagnostics.