A major approach to the investigation of differential gene expression and regulation of protein synthesis in animal cells is through the study of messenger RNA. Recently, intense efforts have centered on the isolation, characterization, and quantitation of the m-RNAs coding for specific proteins. Therefore, the purification method of m-RNA must be improved.
Human Embryonic Kidney(HEK) cells were cultured in MEM media. Total RNA was extracted from HEK cells by CsCl centrifugation method or selective method, then passed through oligo-d(T) cellulose column to obtain total m-RNA. Messenger activity of the preparation was checked in vitro using the rabbit reticulocyte lyste (RRL) system and the peptides synthesized in vitro were separated on SDS-polyacrylamide gel electrophoresis(PAGE). Identification of urokinase(UK)-like material was proved by immunoprecipitation with antibody raised against highly purified UK.
Purity of total RNA prepared by CsCl centrifugation method was 1.6 and maximum absorbance peak appered 260nm. But purity of total RNA prepared by selective method was 1.4 and maximum absorbance peak appeared 270nm. Purity of m-RNA produced by CsCl centrifugation method was 2.0 and maximum absorbance peak appeared 260nm. But purity of m-RNA produced by selective method was 1.69 and maximum absorbance peak appeared 260nm. Quantitation of m-RNA produced by CsCl centrifugation method was 243ug. And quantitation of m-RNA produced by selective method was 177ug.
In conclusion, it was found that CsCl centrifugation method appeared to be superior to the selective method interms of simplicity, processing time, yield, intactness, and biological activity of resulting RNA. And autoradiography showed that m-RNA coded for urokinase-like material.
본 실험은 gene expression 의 분화와 단백질 합성의 조절과 유전 공학적 방법을 이용한 특정 단백질의 다량 생산을 위해 필요로 하는 mRNA purification 의 방법을 개선 하려는데 그 목적이 있다. Total RNA 를 분리하는 방법으로 CsCl centrifugation method 와 selective method 두 가지를 사용 하였고 total RNA 로 부터 m RNA 분리는 oligo (T)-cellulose column 을 사용하였다. 위의 방법을 수행함에 있어 CsCl centrifugation method 가 total RNA 의 순도와 total m-RNA 의 순도와 최종 산물의 양에 있어서 selective method 에 비해 우수함을 발견 하였다. 즉
(1) Total RNA 의 purity 는 CsCl centrifugation method 에서 1.6 이고 selective method 에서 1.4 를 얻었다.
(2) m-RNA 의 purity 는 CsCl centrifugation method 에서 2.0 이고 selective method 에서 1.68를 얻었다.
(3) m-RNA 의 양은 CsCl centrifugation method 에서 243 ug 이고 selective method 에서 177 ug 을 얻었다.
(4) Autoradiography 에서 total m-RNA 중에 유로키나제를 발현하는 m-RNA 가 있음이 확인됨.
