Towards safer stable isotope probing — effect of formamide on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation

Abstract

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.