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Maximizing RNA Extraction From Plants Grown on the International Space Station.  A.J. Stimpson^\, R.S. Pereira, M.J. Correll ,     Dept. of Ag. and Bio. Engineering, Univ. of Florida, Gainesville, FL.

    Due to the significant investments in cost and effort associated with spaceflight experiments, the biological samples that are returned to Earth for genetic analyses are extremely valuable.  In the case of the TROPI experiments (for phototropism and gravitropism experiments) that were performed on the International Space Station in 2006, the amount of material from the Arabidopsis thaliana seedlings was limited due to reduced germination.  To maximize the amount of RNA extracted from the available material, we have compared different tissue disruption methods and protocols using a Pellet Pestle Motor (Kontes, NJ) and the Minibeadbeater-8 (Biospec, OK).  Seven-day-old seedlings were processed from 1 plant (~200mg) to 30 plants (~ 6mg).  The Minibeadbeater-8 samples were removed and placed in 2.0 mL microcentrifuge tubes 2/3 full of 2.3mm Silica/Zirconia beads with the 450 mL of lysis buffer (RLT Buffer, Qiagen, CA) and were run for 3 minutes on the beater at maximum speed.  The Pellet Pestle Motor samples were ground in an eppendorf tube until no visible particulate matter remained (~3 min).  The tests were run both with and without an optional 3 min incubation period at 56°C.  After tissue disruption with both methods, RNA isolation was performed following the protocol of the RNeasy Plant Mini Kit (Qiagen, CA).  Results show that the Minibeadbeater-8 yields consistently higher total RNA than the Pellet Pestle Motor, and that the samples without incubation gave higher total RNA.  Using 5-plant samples, the Minibeadbeater-8 with incubation gave an average of 1.11 mg total RNA, while without incubation yielded an average of 1.38 mg.  The Pellet Pestle Motor gave 0.66 mg and 0.75 mg, respectively. However, after comparing the quality of isolated RNA quality using a Bioanalyzer Pico LabChip (Agilent, CA) the beadbeater resulted in significantly more degradation in RNA, likely due to temperature.  Further testing will determine the effects of temperature on RNA degradation using the bead beater and test the effectiveness of these extraction protocols for microarray analysis.

(Supported By: NASA NCC2-1200)