1、 KSKSKSKSKSKSKSK KSKSKS KSKSK KSKS KSK KS KS P 1018DNA DNA KS P 1018:2012 2012 9 17 http:/www.kats.go.krKS P 1018:2012 : ( ) ( ) ( ) : (http:/www.standard.go.kr) ： ：2012 9 17 2012-0423 ： ： ( 02-509-7294) (http:/www.kats.go.kr). 10 5 , . KS P 1018:2012 i ii 1 1 2 1 3 .1 4 1 4.1 .1 4.2 RNA 1 4.3 RNA .
2、2 5 2 5.1 2 5.2 2 5.3 2 6 2 6.1 RNA .2 6.2 RNA 3 6.3 RNA 3 6.4 RNA .4 7 RNA .4 A( ) RNA .5 A.1 / / 5 A.2 (monophasic) (single-step) 6 A.3 RNA 8 B( ) RNA / 10 B.1 10 B.2 .10 11 KS P 1018:2012 ii . DNA . . , , . . , , . , , . KS P 1018:2012 DNA DNA The technology for gene expression analysis using DNA
3、 microarraysMethods for isolation of genomic samples and quality control 1 DNA . 2 . . ( ) . KS A ISO Guide 30, KS J ISO 21571, KS P 1017, DNA 3 KS P 1017 . 4 4.1 DNA DNA ( RNA) . RNA RNA , 1 , . RNA (RNase) . RNA 70 . 4.2 RNA KS P 1018:2012 2 RNA ( ) RNA , DNA RNA . RNA A , . 4.3 RNA RNA DNA . RNA
4、 ), ( ) . , . B RNA . 5 5.1 RNA KS P 1017, 5.1.1 , RNA ( , ) . 5.2 KS P 1017, 5.1.2 . 5.3 KS P 1017, 5.1.3 . 6 6.1 RNA 6.1.1 RNA , RNA . , RNA . , 70 RNA . 70 . RNA . KS P 1018:2012 3 6.1.2 ( , ) KS J ISO 21571, 5.1.2 . RNA . , . ( ) . , , . 6.2 RNA 6.2.1 RNA . , RNA DNA(genomic DNA) , ( ) . , / .
5、 RNA / . RNA 70 . RNA A . RNA . RNA RNA , 70 , . 6.3 RNA 6.3.1 RNA , , . , RNA . RNA RNA , . 260 nm . B . KS P 1018:2012 4 6.3.2 . , . , . / (KS A ISO Guide 30). 6.3.3 RNA RNA 260 nm /230 nm, 260 nm /280 nm , RNA (integrity) RNA(rRNA) . 6.4 RNA RNA ( , 70 ) . RNA RNA 1 . RNA RNA . 7 RNA RNA . , , (
6、 ) RNA RNA KS P 1018:2012 5 A ( ) RNA A.1 / / A.1.1 ( ) / / A.1.1.1 ( 1) RNA . A.1.1.2 20 , . A.1.1.3 (guanidinium thiocyanate) (denaturant) / . DNA , . . , / / DNA RNA . A.1.1.4 . A.1.1.5 A.1.1.5.1 (ethanol), 99 % . A.1.1.5.2 (chloroform) A.1.1.5.3 (isoamyl alcohol) A.1.1.5.4 (isopropanol) A.1.1.5.
7、5 (equilibrated phenol), pH7.8, SDS . KS P 1018:2012 6 A.1.1.5.6 PBS (phosphate-buffered saline) A.1.1.5.7 (sodium acetate), 2 M, pH 4.0 A.1.1.5.8 (glacial acetic acid) A.1.1.5.9 TE , 0.01 M , 0.001 M EDTA, pH 8.0 . A.1.1.5.10 D (solution D), 4 M , 0.025 M , 0.5 % , 0.1 M - A.1.1.6 A.1.1.6.1 , 4 , 1
8、0 000g A.1.1.6.2 , 25 70 A.1.1.6.3 ( ) A.1.1.6.4 ( ) A.1.1.6.5 , A.1.1.6.6 A.1.1.7 A.1.1.7.1 RNA ( 3, 4). , / ( ) . ( 5, 6). A.1.1.7.2 . 100 mg 3 mL D , 1106 2 mL D . ( D 1 mL ) 0.1 mL 2M (pH 4.0), 1 mL , 0.2 mL - 10 15 , 4 20 10 000g . 20 1 , 4 30 10 000g RNA . 2 70 % , . RNA . A.2 (monophasic) (si
9、ngle-step) A.2.1 KS P 1018:2012 7 ( 2) DNA, RNA, . RNA RNA ( 7, 8). A.2.2 20 , . . A.2.3 A.1.1.3 . , . . A.2.4 . A.2.5 A.2.5.1 (ethanol), 99 % . A.2.5.2 (chloroform) A.2.5.3 (isopropanol) A.2.5.4 , . . A.2.5.5 TE , 0.01 M , 0.001 M EDTA, pH 8.0 . A.2.6 A.2.6.1 , 4 , 10 000g A.2.6.2 , 25 70 A.2.6.3 (
10、 ) A.2.6.4 ( ) A.2.6.5 , A.2.6.6 A.2.7 A.2.7.1 KS P 1018:2012 8 RNA . , / ( ) . . A.2.7.2 . ( 1107 1 mL, 100 mg 1 mL ), 5 . 0.2 mL(1 mL ) , 5 (phase) . 4 , 12 000g 10 , . 0.5 mL 4 , 12 000g 10 . RNA 2 70 % , . RNA . A.3 RNA A.3.1 RNA . . (CsCl) . RNA . A.3.2 . . . A.3.3 RNA (silica-gel) . , , . (sal
11、t) RNA . RNA . RNA . A.3.4 . A.3.5 A.3.5.1 (lysis buffer), KS P 1018:2012 9 A.3.5.2 (wash buffer), A.3.5.3 RPE (buffer), A.3.5.4 - (-mercaptoethanol) A.3.5.5 TE , 0.01 M , 0.001 M EDTA, pH 8.0 . A.3.5.6 (ethanol), 99 % . A.3.6 A.3.6.1 , 4 , 10 000g A.3.6.2 , 25 70 A.3.6.3 ( ) A.3.6.4 ( ) A.3.6.5 , A
12、3.6.6 A.3.7 A.3.7.1 RNA . , / ( ) . . A.3.7.2 . ( 1107 0.6 mL, 30 mg 0.6 mL ), . 3 12 000g , 70 % . (spin column) 15 8 000g . , 0.7 mL , 15 8 000g . , RPE 0.5 mL , 15 8 000g (2 ). 2 8 000g . TE RNA . KS P 1018:2012 10 B ( ) RNA / B.1 B.1.1 RNA . KS J ISO 21571 . RNA 260 nm 1 37 g/mL . , 230 nm, 260
13、 nm, 280 nm , . , 260 nm/230 nm RNA , 1.8 . 260 nm/280 nm , 1.8 2.0 . B.2 B.2.1 RNA . Agilent Technologies 2100 Bioanalyzer . . . KS P 1018:2012 11 1 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 19
14、87, vol. 162, p. 156 159 2 Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques, 1993, vol. 15, p. 532 534 3 Puskas LG, Zvara A, Hackler L Jr, Micsik T, van Hummelen P. Production of bulk amounts of universal RNA for
15、 DNA microarrays. Biotechniques, October 2002, vol. 33, no. 4, p. 898 900, 902, 904 4 Ojaniemi H, Evengard B, Lee DR, Unger ER, Vernon SD. Impact of RNA extraction from limited samples on microarray results. Biotechniques, November 2003, vol. 35, no. 5, p. 968 973 5 Simmons CA, Zilberberg J, Davies
16、PF. A rapid, reliable method to isolate high quality endothelial RNA from small spatially-defined locations. Annals of Biomedical Engineering, October 2004, vol. 32, no. 10, p.1453 1459 6 Scholmm T, Luebke AM, Sultmann H, Hellwinkel OJ, Sauer U, Poustka A, David KA, Chun FK, Haese A, Graefen M, Erbe
17、rsdobler A, Huland H. Extraction and processing of high quality RNA from impalpable and macroscopically invisible prostate cancer for microarray gene expression analysis. International Journal of Oncology, September 2005, vol. 27, no. 3, p. 713 720 7 Xiang X, Qiu D, Hegele RD, Tan WC. Comparison of
18、different methods of total RNA extraction for viral detection in sputum. Journal of Virological Methods, May 2001, vol. 94, no. 1 2, p.129 135 8 Barbaric D, Dalla-Pozza L, Byrne JA. A reliable method for total RNA extraction from frozen Human bone marrow samples taken at diagnosis of acute leukaemia. Journal of Clinical Pathology, November 2002, vol. 55, no. 11, p. 865 867 DNA DNA 153787 1 92 3(13) (02)26240114 (02)262401489 http:/ Korean Agency for Technology and Standards http:/www.kats.go.kr KS P 1018:2012 KSKSKS SKSKS KSKS SKS KS S
