Pellet leucocytic cells from a 50 ml culture flask. (1x105-2x106 cells/ml) in a 50 ml Falcon tube. Pour off medium, turn Falcon tube upside down and put it on a ...
%.) 1993 Oxford University Press
4852 -4853 Nucleic Acids Research, 1993, Vol. 21, No. 20
Simple method of RNA isolation from human leucocytic cell lines Franz Vauti and Wolfgang Siess Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Pettenkoferstral3e 9, 80336 Munchen, Germany Received July 19, 1993; Revised and Accepted September 6, 1993 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 (>107) 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 106 cells: RNA yield 30-50 jig) and large scale (1 x 108 cells: RNA yield 300-600 ILg) 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 (1x105-2x106 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 1l 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 y1 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 ,I DEPC-treated water and transfer suspension to another tube. Then add 700 itl acidic phenol (pH 4.3) and vortex vigorously. Add 140 td chloroform/isoamyl-alcohol (24:1) and vortex vigorously. Spin tube (13000 g, 10 min., 4°C) and transfer 650 $1 from the aqueous upper phase into another tube and vortex
vigorously with 650 Al chloroform/isoamyl alcohol (24:1). Spin again (13000 g, 10 min, 40C) and carefully recover 500 1d from the aqueous supernatant and transfer it into a new tube. Add 50 11 8 M LiCl, mix, add 500 A1 precooled (-200C) isopropanol and mix well (do not vortex). Precipitate RNA for 30 min. at -200C. Spin down RNA at 11000 xg, 10 min., 4C and remove supernatant completely. Rinse pellet with 1 ml 70% ethanol and spin again at 13000 xg, 5 min., 40C. Aspirate supernatant, dry the pellet only slightly and resuspend RNA in DEPC-treated water (100,ul). Measure O.D. and store RNA suspension at- 20°C. A wide range of cell numbers (5 x 106- 108 cells) was lysed in always the same volume of solution D (600 IAI), instead of adjusting the volume of solution D to the actual cell count (100 /d solution D per 106 cells) as indicated in (2). This procedure dramatically reduced the volume of solution D (up to 20-fold)
Figure 1. RNA-Blot. RNA from U937 cells was isolated. Increasing amounts of total RNA (5 yg, 10 sg, 15 ltg, 20 jig, 25 jLg) were dried in a speed vac. RNA-pellets were dissolved by adding 20 yd of RNA-sample buffer [1 ml freshly prepared RNA-sample buffer contained: 212 p1 DEPC-treated H20; 100 Ad 10x RNA-running buffer (0.2 M MOPS, 50 mM sodium-aet pH 5.3; 5 mM EDTA pH8.0); 175 yd formaldehyde; 500 ul formamide; 3 gl ethidium-bromide (10 mg/ml); 10 Al 10% SDS]. The tubes were shaken on an Eppendorf thermomixer at 65°C for 15 minutes and chilled in icewater. 5 jl RNA-loading buffer (15% Ficoll; 0.5% bromophenolblue) was added and the samples were loaded on a gel (1.2% agarose; l x RNA-nnning buffer; 0.66 M formaldehyde) with narrow slots (I x 10 mm). RNA in the samples was allowed to settle for 15 minutes before the run (20 V; 16 hours) was started. After separation (gel electrophoresis apparatus GNA200, Pharmacia) RNA was completely transferred onto Nytran nylon membrane (Schleicher and Schuell) by vacuum blotting (Pharmacia) for two hours. The RNA was fixed on the membrane by UV autocrosslinking (Stratagene). The blot was shortly rinsed in distilled water and washed with 2 x SSC, 0.1% SDS at room temperature for about 15 minutes.
Nucleic Acids Research, 1993, Vol. 21, No. 20 4853 for high cell numbers, but resulted in a very viscous lysate. The RNA extractions following the original protocol (2) were then difficult to perform and resulted in impure RNA (Figure 3). Especially phenol/chloroform extractions led to a huge DNA and protein cushion at the interphase that occupied also the aqueous upper phase, making it impossible to recover RNA without contaminants. We simply overcame this problem by initially shearing the cellular DNA with a 23G needle to reduce the viscosity of the lysate (additionally shearing with a 27G needle improves the result). The less viscous lysate was then precipitated with LiCl, resulting in a still impure initial RNA pellet. However, the majority of the contaminants were discarded by aspiration of the supematant. This step facilitated the subsequent RNA isolation (see above) very efficiently. Different amounts of total RNA (Figure 1) or total RNA isolated from different sources (Figure 2A) were separated by agarose gel electrophoresis. As can be seen in Figure 1, small as well as large amounts of RNA give excellent sharp 18S and 28S rRNA bands. No DNA contmination is visible. Our method produced excellent RNA isolates from various leucocytic cell lines
(Figure 2A). The RNA extracts were further used for measuring mRNA of N-ras, a gene with low expression (Figure 2B) and of (3-actin, a gene with high expression Figure 2C). ACKNOWLEDGEMENTS The study was supported by the Bundesministerium for Forschung und Technologie, Germany (07 ERG 03-7) and August-Lenz-Stiftung. REFERENCES Chirgwin,J.M., Przybyla,A.E., MacDonald,R.J. and Rutter,W.J. (1979) Biochemistry 18, 5294-5299. 2. Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem. 162, 156-159. 1.
3. Bimnboim,H.C. (1988) Nucleic Acids Res. 16, 1487-1497.
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Fiue 3. Agarose gel eletrporesis of RNA isolated from undifferentiated U937 cells according to the original protocol of Chomczynski and Sacchi (2). Cells (2 x 10) were lysed in 600 ji1 solution D and RNA was isolated exactly as described in (2). RNA isolates (10 jig) from two independent experiments were fractionated as described in the legend of Figure 1. DNA contamination and poor RNA separation are visible.
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Figure 2. A) Agarose gel electrophoresis of RNA from the human promonocytic cell lines U937 and MM6, the promyelocytic cell line HL60 and the human eryroleukemic (HEL) cell line. 10 jg of total RNA were fractionated as descaibed in legend to Figure 1. B) Northern blot analysis of N-ras mRNA, a low expression gene. The RNA-blot, obtained from the agarose gel shown in (A) was prehybridized in 5 ml QuickHyb hybridization solution (Stratagene) for 20 minutes at 680C. A 32P-labeled, heat denatured human N-ras probe was added to the hybridization solution at 5 x 106 cpm/ml. The hybridization was performed one hour at 68°C in a hybridization oven (HybAid). The membrane was washed 3 times with 0.1 x SSC, 0.1 % SDS (20 min each) at 600C and exposed to Kodak X-OMAT AR film at -800C for 12 hours with two intensifying screens. C) Northern blot analysis of the 3-aciin mRNA. The hybridization and washing was performed as described (B) after stripping the N-ras probe. A 32P-labeled human actin probe was used (1 x 106 cpm/ml); film exposure was only 1 hour.
