The In-Situ mRNA Sample Homogenizer
To date, studying gene expression has been practically impossible to do at depth, as the organisms alter their transcriptomic RNA expression quickly during recovery to the surface. For very small organisms, such as microbes or meiofauna, there are some remarkable tools that enable one to study gene expression in situ. Also, several groups have used RNA preservatives to reasonably halt transcription and stabilize RNA for analyses. With larger invertebrates or other animals, however, the relative rate of preservative diffusion through tissues is much slower. In these organisms, dropping them into preservative will likely cause dramatic shifts in expression before transcription is halted, resulting in artifacts in transcription. To enable effective analyses of gene expression in freshly collected organisms, we developed an in situ sample homogenizer that macerates and preserves organisms in RNA fixative in situ. The in-Situ mRNA Sample Homogenizer (or iSMASH) is a hydraulically-powered custom blender systems that quickly homogenizes samples in an RNA preservative solution at the seafloor. The entire process takes very little time, one the order of three to five minutes, yielding high-quality transcriptomic data that are likely to be much more representative of the conditions experienced by the organisms in nature.
To examine gene expression in situ, we have recovered individual snails from chemically distinct microhabitats in the Lau basin and vent tubeworms from the East Pacific Rise, preserved them in situ using the iSMASH, and isolated host mRNA and generate cDNA as previously described. This cDNA will be used in conjunction with cDNA pools from shipboard experiments (which will be the tester or driver as appropriate) to recover those genes being expressed by organisms in situ. While in situ gene expression is not intended to be a quantitative proxy for metabolic rate, this approach may prove to be the best means of examining physiological poise in deep-sea animals. This proposal provides a unique opportunity to develop such exciting approaches because all the technologies necessary (shipboard pressurized respirometry, in situ voltammetric chemistry, and ecologically relevant sampling strategies) are employed in this research.
Open Source Information on iSMASH
Figure 1. A schematic of how the iSMASH is configured on the “porch” of a submersible or ROV. The magnetically latched lids are removed and a sample is dropped in. The vessel is rapidly flushed with preservative, displacing the seawtaer inside. Within minutes, hydraulics are used to actuate the iSMASH blades and homogenize the sample.
Figure 2. A photograph of the iSMASH prior to deployment on the ROV Jason submersible during our expedition to the Lau Basin in 2009.