Yi Sun, Ying-Wei Mao, Wen -Qin Song, Rui-Yang Chen
The GTLES method for high purified and high integrative RNA isolated from Artemia was established. Comparing with other methods that involved guanidinium thiocyanate, this method costs less and produces RNA molecules with better quality. The working concentration of guanidinium thiocyanate was reduced more than 40 times compared with previous methods. The isolated RNA with this method gave 3 bands (28S, 18S, 5S) when analyzed by 1.1% agarose gel electrophoresis and contained less contaminating proteins as examined by the O.D.260/280 ratio (>2.0).
The following protocol describes isolation of RNA from 200mg of adult Artemia. Living Artemia was ground in cool mortar with liquid nitrogen into a fine powder. 2ml of GTLES buffer (0.1M guanidinium thiocyanate, 0.2M Tris-HCl, 0.005M EDTA (pH 8.0), 1% sarcosyl, 0.1M LiCl, which was stored at 4 and 7.2 microl beta-mercaptoethanol was added per ml GTLES buffer before use) was added into the mortar and the mixture was homogenized until defecation. Then it was transferred to a 7-ml tube. 200microl of 2M sodium acetate (pH4.0), 2ml of phenol (water saturated) and 400microl of chloroform-isoamyl alcohol mixture (49:1) were added. The sample was cooled on ice for 20 min, then centrifuged at 11,000 rpm for 20min at 4 and extracted again at the same condition. The aqueous phase was transferred to a fresh tube and RNA was precipitated by the same volume of isopropanol at -20 for at least one hour. After centrifugation at 12,000rpm for 15min at 4, the RNA pellet was rinsed with 400microl of 3M sodium acetate (pH 5.2) twice and 75% ethanol twice. The RNA pellet was vacuum dried and dissolved in 50 microl H2O.
The RNA preparation could be used for almost all kind of downstream applications such as cDNA synthesis, northern blot or RT-PCR.
(correspondence from SunYi, Department of Biology, Nankai University, Tianjin, China)