A rapid filtration apparatus for harvesting cells under controlled conditions for use in genome-wide temporal profiling studies

Patricia V. Burke, Liang Chuan Lai, Kurt E. Kwast

Research output: Contribution to journalArticlepeer-review

Abstract

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 1.2μm cellulose acetate filters for isolating RNA for genome-wide temporal profiling analyses.

Original languageEnglish (US)
Pages (from-to)29-34
Number of pages6
JournalAnalytical Biochemistry
Volume328
Issue number1
DOIs
StatePublished - May 1 2004

Keywords

  • Anaerobic
  • Filtration
  • Gene expression
  • Microarray
  • Yeast

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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