KS P 1018-2012 The technology for gene expression analysis using DNA microarrays－Methods for isolation of genomic samples and quality control《利用DNA微数列的基因发现分析技术 隔离的基因组提取法与质量管理》.pdf

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 .

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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

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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