A rapid filtration apparatus for harvesting cells under controlled conditions for use in genome-wide temporal profiling studies.
Gene expression can respond rapidly to changes in environmental conditions. To effectively monitor these responses, we built a filtration apparatus that allows for the rapid harvesting and processing of moderate volumes of yeast cells under controlled atmospheric conditions (e.g., anaerobic conditions). Harvesting by filtration offers several advantages over that by centrifugation, especially when rapid, repeated sampling of dilute cultures is required. A number of different filter membranes, including cellulose acetate, mixed esters of cellulose, regenerated cellulose, polycarbonate, and polyvinylidene fluoride, were assayed for harvest efficiency and the quality of RNA obtained by hot-phenol extraction from cells directly adhering to the membranes. To determine the suitability of the RNA for microarray analyses, we quantified both cDNA yield from reverse transcription and the indirect coupling of Cyan dyes. In general, filtration times, cell yields, and RNA quality were similar among the filters examined, although some media components (e.g., antifoam) can cause fouling of smaller-pore-sized filters. Thus, choice of a membrane will depend on the particular medium, ease of filter handling, or on other experimental considerations. We routinely use this filtration apparatus with Osmonics 1.2 microm cellulose acetate filters for isolating RNA for genome-wide temporal profiling analyses.
Burke
Patricia V
PV
Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL 61801, USA.
Lai
Liang-Chuan
LC
Kwast
Kurt E
KE
D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D019411:Clinical Laboratory Techniques / Q000295:instrumentation / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000032:analysis; D013997:Time Factors; D015003:Yeasts / Q000502:physiology
D012313:RNA