Simple method of RNA isolation from human leucocytic cell lines.

Very reliable methods have been developed to extract RNA from different tissues and cell lines (1, 2, 3). RNA preparation, however, is still cumbersome and time consuming, especially when extracting a large number of cells (>10) and cell samples. The isolation of RNA from leucocytic cells is particularly difficult for three reasons: a) high levels of endogenous RNase; b) low amount of RNA (RNA/DNA ratio of 0.08 in granulocytes compared to 4.0 in cultured fibroblasts) (3); c) contamination of the RNA isolate with DNA and protein. To overcome these problems, we developed a simple and efficient RNA isolation procedure by modifying the method described by Chomczynski and Sacchi (2). Our modified protocol has the following advantages: a) it is simple to perform and needs no major equipment; b) it reduces drastically the volume of costly and hazardous solvents; c) both, small scale (5 x 10 cells: RNA yield 30-50 /tg) and large scale ( lxlO 8 cells: RNA yield 300-600 jig) RNA extractions can be performed using exactly the same protocol in small 1.5 ml microfuge tubes with many cell samples (up to 20) in parallel in a short time (5 hours); d) the protocol produces extremely pure total RNA at high yield. We used the method mainly to measure quantitavely low level gene expression by Northern blot. The following detailed stepby-step protocol was tested using various undifferentiated and differentiated leucocytic cell lines (U937, HL60, HEL, MM6), but should be applicable to any cell type growing in suspension. Pellet leucocytic cells from a 50 ml culture flask ( l x l 0 5 2 x l 0 6 cells/ml) in a 50 ml Falcon tube. Pour off medium, turn Falcon tube upside down and put it on a kimwipe to drain the pellet. While vortexing the pellet, splash 600 /tl solution D (4M guanidinium thiocyanate; 25 mM sodium citrate, pH 7; 0.5% sarcosyl; 0.1% 2-mercaptoethanol; 0.1% antifoam A) to the cells. Vortex until reaching a clear very viscous lysate. Pour the suspension directly into a 1.5 ml Eppendorf cup. Aspirate the lysate into a 2 ml syringe fitted with a 23G-needle and expel it into the tube applying high pressure. Repeat this step 10 times. (Antifoam A prevents foaming while shearing DNA). Add 600 /tl 8 M LiCl, mix by turning the cup until the clear suspension becomes cloudy (do not vortex). Cool on ice for at least 1 hour. Spin down precipitated RNA at 13000xg for 20 min. at 4°C. Remove completely supernatant. Take up RNA pellet in 650 /tl DEPC-treated water and transfer suspension to another tube. Then add 700 /tl acidic phenol (pH 4.3) and vortex vigorously. Add 140 jtl chloroform/isoamyl-alcohol (24:1) and vortex vigorously. Spin tube (13000 g, 10 min., 4°C) and transfer 650 /tl from the aqueous upper phase into another tube and vortex vigorously with 650 /tl chloroform/isoamyl alcohol (24:1). Spin again (13000 g, 10 min, 4°C) and carefully recover 500 /tl from the aqueous supernatant and transfer it into a new tube. Add 50 III 8 M LiCl, mix, add 500 /tl precooled (-20°C) isopropanol and mix well (do not vortex). Precipitate RNA for 30 min. at -20°C. Spin down RNA at HOOOXg, 10 min., 4°C and remove supernatant completely. Rinse pellet with 1 ml 70% ethanol and spin again at 13000Xg, 5 min., 4°C. Aspirate supernatant, dry the pellet only slightly and resuspend RNA in DEPC-treated water (100 /tl). Measure O.D. and store RNA suspension a t 20°C. A wide range of cell numbers (5 X10-10 cells) was lysed in always the same volume of solution D (600 /tl), instead of adjusting the volume of solution D to the actual cell count (100 /tl solution D per 10 cells) as indicated in (2). This procedure dramatically reduced the volume of solution D (up to 20-fold)