Washed Away; How Not to Lose Your RNA during Isolation

<i>Cryptosporidium</i> oocysts have extremely robust walls that protect the parasite against environmental pressures. Analyses must be sensitive to detect the few organisms (if any) present in environmental samples. After a series of negative nucleic acid amplification results on spiked samples, following a standard RNA isolation protocol, it seemed probable that oocyst RNA had been lost in the washing steps of the isolation protocol. By reducing both the volume of wash buffer and the number of washing steps, positive results could be re-established. Insufficient washing, however, seemed to prevent downstream analysis, probably because of inhibitory substances remaining in the RNA isolate. Nucleic acid isolation protocols for low numbers of "difficult" organisms should be adapted, according to the material to optimize the balance between removal of inhibitors and retention of target, thereby improving the performance of the technique.