Use of an armored RNA standard to measure microcystin synthetase E gene expression in toxic Microcystis sp. by reverse-transcription QPCR
Limnol. Oceanogr. Methods 7:509-520 (2009) | DOI: 10.4319/lom.2009.7.509
ABSTRACT: Microcystin is a secondary metabolic peptide toxin known to cause hepatotoxicosis and carcinogenicity in vertebrates. It is produced by various bloom-forming cyanobacterial species constituting a serious threat to the quality of freshwater reservoirs worldwide. There is an intense interest to understand the biological function of microcystin and the regulatory mechanism behind its biosynthesis in order to develop strategies for freshwater management. To date, changes in toxin production in response to various environmental parameters have been studied using direct toxin analysis. Despite extensive research over the past two decades, the function of microcystin remains poorly understood. In this study, we developed an alternative method to study the regulation of microcystin production in toxic Microcystis sp. (and alternatively in Anabaena sp.) at the level of gene expression targeting microcystin synthetase E (mcyE) transcripts. We developed a durable and ribonuclease-resistant armored RNA standard to monitor for the integrity of all nucleic acid processing steps including extraction, reverse transcription, amplification, and detection. The armored RNA was designed to consist of the mcyE target matrix with a unique internal probe-binding sequence. To validate the efficacy of the method, we conducted a comprehensive survey of mcyE expression profiling in batch cultures of Microcystis sp. during exponential, linear, and stationary growth phase. Our transcriptional analysis indicates a growth-phasedependent expression of mcyE, with the maximal level of expression observed during mid-log growth. With progressing age of the cultures, mcyE was gradually down-regulated, with expression levels having declined by more than three orders of magnitude during the stationary growth phase.