Bead assays are an important rapid microbial detection technology suitable for extremely low pathogen levels. We report a bead assay for rRNA extracted from Escherichia coli K12 that does not require amplification steps and has readout on an Agilent 2100 Bioanalyzer flow cytometry system. Our assay was able to detect 125 ng of RNA, which is 16 times less than reported earlier. The specificity was extremely high, with no binding to a negative control organism (Bacillus subtilis). We discuss challenges faced during optimization of the key assay components, such as varying amounts of RNA in the samples, number of beads, aggregation, and reproducibility.