Cesium chloride gradient centrifugation improves the quality of total RNA preparations from the gastropod Strombus gigas and the coral Montastraea faveolata

RNA preparation from marine invertebrate samples including gastropods and cnidarians is often difficult, producing low-purity RNA samples that perform poorly in downstream procedures. This is increasingly problematic given the current interest in transcriptional studies of marine invertebrates. We report an RNA preparation method using a phenol/chloroform extraction followed by CsCl gradient centrifugation, modified from previous reports and adapted for a modern benchtop ultracentrifuge. This method produced improved spectrophotometric indicators of RNA quality (A260/A280 and A260/A230) for Strombus gigas Linnaeus, 1758, testis samples and improved RNA integrity number (RIN) for Montastraea faveolata Ellis, 1786, samples, relative to a preparation procedure that relies solely on chemical extraction of RNA. Rate of success in microarray labeling reactions was slightly, but not significantly, improved following CsCl gradient centrifugation. A real-time RT-PCR assay performed using CsCl-prepared samples was efficient (92.5%) and produced consistent and reproducible results.

Research highlights► We present a CsCl-based method for S. gigas and M. faveolata RNA preparation. ► Method produces intact RNA more pure than RNA prepared without a CsCl gradient. ► Resulting RNA can be labeled with Cyanine 3 with a high rate of success. ► Resulting RNA performs consistently in real-time RT-PCR.