KS P 1019-2012 The technology for gene expression analysis using DNA microarrays-Experimental procedures for DNA microarrays with direct labelling and measurement of gene expressio.pdf

1、 KSKSKSKSKSKSKSK KSKSKS KSKSK KSKS KSK KS KS P 1019DNA DNA KS P 1019:2012 2012 9 17 http:/www.kats.go.krKS P 1019:2012 : ( ) ( ) ( ) : (http:/www.standard.go.kr) ： ：2012 9 17 2012-0423 ： ： ( 02-509-7294) (http:/www.kats.go.kr). 10 5 , . KS P 1019:2012 i ii 1 1 2 1 3 .1 4 DNA 2 4.1 .2 4.2 2 4.3 .3 4.

2、4 4 5 4 5.1 4 5.2 DNA .4 5.3 5 5.4 .5 5.5 .7 5.6 .7 5.7 8 5.8 XML 8 A( ) DNA 9 A.1 .9 A.2 .9 A.3 9 B( ) .13 B.1 (gridding) (spot finding) .13 B.2 (segmentation).13 B.3 (quantification).13 C( ) .14 C.1 .14 C.2 14 D( ) .16 D.1 .16 D.2 .16 E( ) KSMGED-ML(Korean Standards of Microarray Gene Expression D

3、ata Markup Language) version 1.0 KSMGED-DTD(Korean Standards of Microarray Gene Expression Data Document Type Definitions) version 1.0 .17 21 KS P 1019:2012 ii . . , , , , . , DNA , , . DNA . DNA DNA . DNA Cy5 Cy3 2 , . , ( , 1 ) , , . . , , . , , . KS P 1019:2012 DNA DNA The technology for gene exp

4、ression analysis using DNA microarrays Experimental procedures for DNA microarrays with direct labelling and measurement of gene expression 1 DNA DNA . DNA RNA , DNA . 2 . . ( ) . KS J 0009, (PCR) KS P 1017, DNA MGED Ontology (http:/ , . MGED Ontology MGED Society (http:/www.mged.org/) , , . MGED On

5、tology version 1.3.1.1 , MO_XXX MGED Ontology . 3 KS P 1017 . KS P 1019:2012 2 4 DNA 4.1 4.1.1 DNA RNA DNA . DNA ( RNA) DNA . 1) RNA cDNA , 2) cDNA , 3) cDNA , 4) . DNA A , . 4.1.2 RNA cDNA RNA mRNA , cDNA . / (exo-/endonuclease) . cDNA . , RNA . 4.1.3 cDNA DNA 1) , 2) (emission) , 3) . , . . 4.1.4

6、DNA cDNA DNA . 1) , 2) , 3) . A . 4.1.5 DNA DNA . . . (photobleaching) . 4.2 4.2.1 KS P 1017, 5.1.1 , DNA , ( , ) KS P 1019:2012 3 . 4.2.2 KS P 1017, 5.1.2 . 4.2.3 KS P 1017, 5.1.3 . DNA . a) b) DNA c) d) e) DNA , 4.3 4.3.1 DNA RNA 20 . , , dNTP, dT(oligo dT), , 20 . ( , , SSC, SDS ) . 4.3.2 RNA cDN

7、A KS J 0009(9.7 ) PCR . RNA cDNA mRNA dT , . RNA , dNTP cDNA . dNTP dNTP (molar ratio) dATP, dCTP, dGTP, dTTP . . , , . RNA DNA 50 g 100 g RNA(mRNA 1 g 2 g ) . , . 4.3.3 DNA KS P 1019:2012 4 cDNA DNA . DNA . cDNA 1 . , (carrier) DNA (ssDNA, yeast tRNA, poly A DNA ) . , . . . 4.3.4 DNA DNA . . A . .

8、DNA . DNA (photobleaching) . 4.4 DNA . RNA , , DNA , , cDNA 5 5.1 KS P 1017, 5.2.1 . 5.2 DNA DNA ( 1 ). KS P 1019:2012 5 1 DNA 5.3 DNA . mRNA 2 , . DNA . , . B (gridding) , (segmentation) (quantification) . Image analysis MGED Ontology “ExperimentalProtocolType”(MO_75) , “feaure_extraction”(MO_928)

9、. 5.4 DNA , (noise) . (Image Analysis) (Spot Quality Control) - (Spot size) - (Signal to Noise Ratio) - (Saturation) (Gridding) (Spot finding) (Segmentation) (Quantification) (Spot filtering) (Between chip Normalization) (Gene Filtering) (Within chip Normalization) KS P 1019:2012 6 . . Spot Quality

10、Control MGED Ontology “QualityControlDescription”(MO_88) “QualityControlDescriptionType”(MO_121) . 5.4.1 . pixel|pixelpixel|exp00sizeFFFq= Fpixel ： Fpixel0 ： 5.4.2 (foreground) (background) . (non-specific binding) . )meanmean(meannoisesigBFFq+= Fmean： Bmean： 5.4.3 , . %10_,0%10_,1sat=pixelssaturate

11、difpixelssaturatedifq 5.4.4 (quality score) . satbkg2bkg1noisesigsizecom41)( qqqqqq =)mean0bkg/()0bkg/max(12),mean0bkg/()0bkg(2)mean/BSD/max(11),mean/BSD/(1bkg2bkg1BfBfqBfBfq+=+=KS P 1019:2012 7 BSD： bkg0： 5.4.5 DNA , C . 5.5 , . 1 . , , . 2 (filtering) MGED Ontology “DataTransformationProtocolType”

12、(MO_165) , “flag_filter”(MO_124) . DNA . 5.5.1 . . 5.5.2 , . 20 % . 5.6 DNA (bias) . (normalization) MGED Ontology “DataTransformationProtocolType”(MO_165) . a) . b) , . KS P 1019:2012 8 c) (pin-group) DNA . d) (slide heterogeneity) . DNA . D . 5.7 , . 5.8 XML E , , KSMGED-ML . KSMGED-ML v1.0 XML 1.

13、0(KS X 6030) , . XML( ) (Standard generalized markup language ISO 8879) . W3C XML , http:/www.w3c.org/XML . XML , , KSMGED-ML , . KSMGED-ML v.1.0 KSMGED-DTD v.1.0 . KSMGED-ML KSMGED-DTD . KSMGED-ML KSMGED-DTD MGED society MAGE-OM(http:/www.mged. org/Workgroups/MAGE/mage-om.html), MAGE-ML(http:/www.m

14、ged.org/Workgroups/MAGE/ mage-ml.html), MAGE-TAB(http:/www.mged.org/mage-tab/) . KS P 1019:2012 9 A ( ) DNA A.1 50 g RNA DNA . RNA DNA . , . A.2 10 DNA . A.3 A.3.1 A.3.1.1 A.3.1.1.1 dT(2 g/L), dTTP 16 mer . A.3.1.1.2 MML-V (Moloney murine leukemia virus reverse transcriptase, 200 units/L) A.3.1.1.3

15、dNTPs, dATP, dCTP, dGTP 25 mM, dTTP 15 mM A.3.1.1.4 RNase (RNase inhibitor, 5 units/L) A.3.1.1.5 Cy5-dUTP(10 mM) A.3.1.1.6 Cy3-dUTP(10 mM) A.3.1.1.7 (buffer), . A.3.1.1.8 0.1 N NaOH A.3.1.1.9 0.1 N HCl A.3.1.1.10 PCR , . A.3.1.2 A.3.1.2.1 , 10 000g KS P 1019:2012 10 A.3.1.2.2 , 25 70 . A.3.1.2.3 , A.3.1.3 A.3.1.3.1 RNA dT mRNA cDNA . (3). cDNA PCR . (4), (5). , (7). A.3.1.3.2 RNA(50 g ) 4 g dT 65 10 . 2 (10 , dNTP, RNase , Cy5-dUTP, Cy3-dUTP, 400 uni