Rapid method of total RNA mini-preparation from eucaryotic cells.

There have been described a wide variety of methods for the preparation of total RNA from eucaryotic cells (1, 2). These methods are mainly designed for the isolation of RNA from relatively large amounts of cells. In a situation of screening a large number of cell clones, minimized cell cultivation effort and preparation time for total RNA isolation will be of great advantage. We developed a method which fulfils the following criteria: (i) the procedure is very simple, (ii) includes short incubation and reaction times, (iii) needs relatively small amounts of cells and (IV) can be carried out for a great number of cell clones as a multi-sample preparation. We used the rapid method mainly to screen human tumour cell clones for their expression of foreign genes after transfection or transduction of various vector constructs. The brief protocol is the following: Individual clones were seeded into 12 well dishes. After 24 days cultures reached confluence and contained approximately 5 X10 cells per well. For the RNA preparation the medium was discarded and cells were washed once with 1 ml of 0.9% sodium chloride solution. Then 200 jtl of lithium chloride/urea solution (3 M LiCl, 6 M urea) (3) were added to each well and incubated for 5 min at 20°C. This was followed by addition of 1/10 vol. of 3 M sodium acetate solution and of 2.5 vol of absolute ethanol. After mixing the suspension was transferred into an 1.5 ml Eppendorf tube, left for 15 min at 20°C and centrifuged at 12000 rpm for 5 min. The supernatants were discarded and the pellets were resuspended in 100 /il of TES (10 mM Tris pH 7.6, 1 mM EDTA, 0.5% SDS; autoclaved) by vortexing thoroughly. Then 100 til of phenol/chloroform were added followed by vortexing and centrifugation of the probes at 12000 rpm for 5 min. The aqueous phase was transferred into another Eppendorf tube, 1/10 vol. of 3 M sodium acetate and 2.5 vol. of absolute ethanol were added and stored at —70°C for 15—20 min to precipitate the RNA. Probes were then centrifuged at 12000 rpm for 10 min. Pellets were washed once with 1 ml of 70% ethanol, dried and resuspended in 20-40 /A of DEPC-water and stored at -20°C for further investigations. The RNA yields were calculated by OD-260 spectrophotometry and ranged between 10—15 /ig.