tutorial microarray

7/25/2019 Tutorial Microarray

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1. 01

2. 01

3. 013.1. 04

3.1.1. A 04

3.1.2. A 05

3.1.3. A 06

3.1.4. A 09

3.2. 13

4. 13

5. 14

6. 15

7. 16

7.1. 167.2. 17

7.3. 187.4. 19

7.5. 198. A 19

8.1. 20

8.1.1. 208.1.2. 238.2. 248.3. A 248.4. 249. 259.1. 259.1.1. 259.1.2. 269.1.3. 27

9.2. 2810. . 2810.1. 2810.2. A 2810.2.1. 2810.2.2. 3810.2.3. A 4811. 49


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1.

C aa ecee da a ca e ce a c a

ea caa eca, ea, e DNA. O e dfeeca d ceae

fcaee d ee ee ee d de ca e e de ee dee ee.

A caa d e de ee d ee de dfeee ecd de ea a

eede d de fee ecad e a e,

eee aa a deec de ee c ee dfeeca ee ecd e

de e eaad c (caa) de DNA. Ea cca

ee ea e de ee de ae de ee aeaee.

A caacdade de be de dad de ee ca e a caacdade de

aa aaee. O de fae aa a ae de dad ec

e ecde.

2.

Nee a fae a e de eaa ad a eaa da cca de

, dede a fabca d a a ae de ee dfeecaee

ee ad de eae. Paa eaa da ae ae

fae R e ace d B.O be ca fece a e a aa ae de eee de

ad R e Bcdc. Se ada e ecada a a de

cad ecea aa eaa a ae e d bac de dad ea

baae ad a eaa.

3.

Ae da eca de a ea eca ea ae aeaa e

aa faae a a a, ee a ee.

A cca de ee ea e de ee de ae de ee

aeaee, bad caae ee aa de ecd e ef

de ee.

S eaad ae de ee e aa dfeee aeea a

ea ea de dade eeea.


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F 3.1:Iae de .

A

A da ae de ee ca, eee de de e a

acae c, ee:

Deec de f; Reeeca eca;

Geae;

Ecaae eca.

A

A cca de dfee a fa de fa d aea ec aa ed eae d ce eeae aa da. A ee de

cca :

(.a.c);

(.cecece.c);

A: (.e.ae.c);

C A (.affe.c);

A ;

de ea e ;

F

A c d de e fea dfeee cca de ea

dfee a fa de fa d aea ec a a. A fa aba

a cca de fabca de : (a) , (b)

e (c) .

Vee aea a cca (b) .


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F 3.2:Tcca de fabca de . (a) , (b)

, (c) . Fe: Scea e a. (1998).

Sed Le e Pa (2006) aea da eca de cde ee ca

e fa, ad c a eadade da caea:

de cDNA c cDNA cede;

A de ade dedade dd cecaee e c

cede ead.

O c e a d c e ee da caacdade de a

eca ea de cede e ca bda c a eca

ceea e fa a eca da de DNA (Le e Pa, 2006).

F 3.3: DNA.


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3.1.

Sed Eee, 2007, eee de cDNA , ece bacde ce de eca eea (EST). A EST dda aa da ca defae de DNA da fa a a de a eca eea (RNA eae RNA) ac eea. Ee cede eeea d e aca de DNA ceea (cDNA) e e cad e baca. Ac da a cea c a ee d ce e c a ecaeecfca d ee de eee. Ea eca eda d ce,afcada PCR e fada e e eecfca da a, cecda c, aa de aa de e e cada b eecfc.

3.1.1. F DA

: A fae ca de eee ce a ee de

ce de cDNA d de a bac de ce e eaee e

eacad c a e ea eecfc.

A C:C e eee de

de e ad a adade ca de cDNA de

e fcee, e dee e e afcada PCR (Rea e cadea

Peae).

F 3.4:Teccad. Aae ad aa eaa de PCR.

F DA: O fae eecad fad a a de

d b caad Ae e eecfca cecda c

.

O cDNA fad a a caad cDNA da, a c,

cada , a eca de c ee.


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F 3.5:Adead cDNA a a de d.

3.1.2. E A

S ead RNA eae (RNA) RNA a da da ae

ceae de eee (Taae e Ce, ee: ca

caceea e ca a).

A a de cada aa de RNA dd cDNA (DNA ceea)

ac eea. O cDNA de cada aa d c cae

fecee C3e C5 a e ecad c ce de da

dfeee. O cDNA da a ce e d c C3 e

cDNA da a aae e d c C5. O cDNA a de da aa da (a c C3e a c C5)

ad e bdad ca a a de d;

Nee ce a eca de cDNA da da aa, e ca c a

a de d, e aea c a eca ceeae d

cDNA da. Hae ce ee a da aa.

F 3.6: A da aa de cDNA acad ada e ccada a

a aa bda. Fe: S (2008).


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F 3.7:Eaa d ce de bda.

3.1.3. A

De e a a f bdada ea aa a eaa de aae aa ee

ece de aea ec e bd c a da e fea a ea

d e aa daa da ae;

Ee d de :

CCD:Na eca CCD a a ecada c a bacae da a ee e a cea fafa a ae decee da

e de edade eee d (C3 e C5) eee

a (ae ceae de eee) e fa ad aa a

bda (Eee, 2002).

:O a ae fae a aeda a a c

a ce de da eecfc daad a ae

eada.

F 3.8:Lea de e a ae.

O dad b eae de eee de de da ce

ae cca, a aa cada cae, aee a .f.

Peee de ae de ae ab ce a cada a daae e eca ea ae dad e a a ca ae. O A,


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ee, aa aa ae de ae de de DNA, Ee

(1998).

121301G 121301

F 3.9: Iae de eee de . A fa 12130c1G

cede ea d cae ce de da e eca C3e a fa

12130c1R cede ea d ce de da e eca C5.

Ea fa fae ae d eee AA a aad Dd e a.

(2000). O bac de dad dee eee, cd a ae, e de e

://.a.beee.ed/e/e/aa/H/adaa..

F 3.10: de ae de ae de A ead a

a ae ea c da ae 12130c1G e 12130c1R abda da ce

ede aa 12130c1G e eea aa 12130c1R.


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F 3.11:O ee de ae de .

F 3.12:Daaa a da cca de cDNA . Fe: Fa (2007).


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3.1.4. A

A ae ce dad da ae de ee ca;

Vea, ed Eee (2002), a e ad a ae de

ceae de ae de : e de a: Re cada e ;

B: Iae de fd da a (e de e eca

);

Rd: Faa de cb de a ded a ca e e aea

c ea ca fecee;

Aefa: Sa eecfc decee de ea a a bda

eecfca e caa ;

F 3.13: Te ad a ae de ceae de ae de

.

O ceae de ae de de e ddd e ae:

1. Edeeae adeae;

2. Seea d a ( d );

3. Qafca da edade;


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E ;

Nea eaa caa bc e . A dee e fe dadeae:

Bc;

Re de fca d ;

F 3.14:Gadeae d bc fae B.Fe: Eee (2002).

F 3.15:Gadeae e eea d .Fe: Eee (2002).


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( )

Vefca e e eecee a a ;

Md de eea:

Seea de cc f; Seea de cc adaa;

Seea aa;

Seea aa de edade;

F 3.16:Iae de eea cc f e eea aa de

edade eecaee. Fe: Eee (2002).

S fe cc da edade e d.Obee a fa

aba, a edaa da edade de (eea ede) de

d cc, e B a edaa da edade de d (e

fa d cc de d adad). A ea cacada c ed e

de e de d cc ca edade ea a e d e fa d

cc a de d adad. A a edade d cacada :

= ( B )*A.


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F 3.17:Cc da edade d . Fe: Caae (2008).

A a ae da ae de ead a abea ced e

de ee d ee.

F 3.18:Bac de dad de c cd. Ee dad

ae d eee AA a aad Dd e a. (2000). Bac de

dad de e:


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aa cedee f acada c C3 e a a da feca dca e a aacedee f acada c C5.P ee, a fa aba, eee (a) e c de aaa A f acada c C3 (ede) e a aa B f acada c C5 (ee),

eee (b) e d de e a aa A f acada

c C3 e a aa B c C5 ea ed caa f fe e, ea, a aa A f acada c C5 e a aa B c C3.

F 4.1:Ee de deeae aa de da ce.

Na fa aca e:

(a) Deeae c caa dea;

(b) Deeae c Ie de Cae ( );(c) Deeae c Refeca;(d) Deeae e .

O deeae c e de cae ( ) e c be ea cfde ded a efe d cae.N deeae c efeca a aa de eee caadadeaee, aa de a efeca c. Ee de deeae e aaae de de aa dee e adcad eee aaee caada c a aa e aa d caada, baad aa ea efeeca c ada a aa aee. Pde abaaa eee de de a c c e fe deeaec efeca c.

5.

O dad de edade de fecca adcaee aad ad

e fc de de. O fc a e fa fe c dad d eee

A A, Dd e a. (2000).O fc a c :


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C3 C5;

A;

de = 2() 2() e A = 2() + 2().

F 5.1:Gfc de de de C3 C5aa 1

F 5.2:Aaa 1.

Sbe a afa = 2() 2(), A = 2() + 2() acada

e a cca de aa, e bca ed a aa da cca e a

a edade cae ee dfeee bdae.

6. C

Sed Eee, 2007, a edade d bacd ecea aa a ce dae de edade bd. A a aa a a dee dad e fa


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de e e be eaaee ae de d ecad ,eea efe dee d de e ad aa da ce d aede edade da e de a e ae de edade d .A ce d fea bad a edade d bacd daedade d.

7.

Eee de a fe de aa eca a a

de afea a ede d e de ee ca. Paa caa

dee e eda a fe de aa de cada dee.

A cca de aa afae d dad e bca ee ea

fe de aa.A aa de e fea:

De d ;

Ee ;

A Naa de d fea e cada eaadaee e de eed ee d , e eaada ee d . Ee deaa bca ee, ee, d cae de de cada c a ca;A aa ee ada aa e a caa ee deee.

C

Dfeea a efcca da ca d cae;

Dfeea a adade de RNA ca ad aa aca e bda;

Dfeea de ae de ae d de ea da a;

Faa a e da da, ec.

Iec de eae; Pcede de caa e afca adad e ;

7.1. G

fea aa ce de ca. acada ad ee a ea cae(Cy5=k.Cy3).

2 2

2

5/ 3 5/ 35 / 3

i i

i

Log (Cy Cy ) Log (Cy Cy )Log (Cy k.Cy )

=

=


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O ce da db de d a da a da aa e.Te a deaae de cdea efe da db eaca d aa a ee d ee e cdea e a dfeea deedeeda edade de ee.

F 7.1: Pe bea e a ae de . A aca a fa

aaea e a aca de ded. Fe: Eee (2007).

7.2.

Vc deedee da edade de ee a db d a.I de e ad ad e bea MA.

F 7.2:MA c ca de aa .1

Uae d aac aa ee de M e A, caad ( ).Ee d adaa a ca ea de ee caee deada, e ada cca eaca de aa de fc de de. Ta d ea

ea ca eae caae de e dad bead.A a da ee, a ce fea c:


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O ae de C5e C3aad de e bd c:

b(A) ae de aa MA.

7.3.

O ce de dea cede de cDNA eec () () .

F 7.3: eeda e a a ed ea e dea e

caa de de bea de e.

Ee c de aa de e a fe de aa eca de aa e de ee edd.Ee de aa eeae aa deedee da edade deee ed, ea, eaad de e.

F 7.4: G de e de c cedee ca de.

2 2

2

( 3/ 5) ( 5/ 3) ( )

( 5/ ( ). 3)

i i

i

Log Cy Cy Log Cy Cy b A

Log Cy k A Cy

=

=

2 2

2 2

( 5) ( 5/ 3) / 2

e

( 3) ( 5/ 3)

i i

i i

Log Cy A Log Cy Cy

Log Cy A Log Cy Cy

= +

=


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7.4.

Sed Saffd (2008) a aa de d aa ecaa aa a dfeeade ecaa ee aeMde d ee .Ae e a aa d ee dfeecaee ee ee dfeee

aa bac de dad, a, a ecaa de e aeMdee e cae.Se dea Msc da a aad aa ecaa, e Ms = M/s,de M da a de ee ae da aa, es fa de ecaa.O fa de ecaa de e cacad de e cacad ad a (A)e a eaa ba des.E

deMieeea da a de ee ae da aa, e fade ecaasiaa de e ead c:

7.5.

Ea aa ada aa e a caa ee deee. acada a a aa de d aa ce defe de ecaa ee .O e d ad aa a ce de ecaa de d de edeaee eedd aa ea a.

8. A B D

E

Nea eaa, e e dfeee abdae aeca e eaca,

deeded da ee bca eda c eee.O a c de ae de :

a) Bca de ee dfeecaee ee (DE);

b) C de aae (a aa ee c aa aa);

c) Busca de de ee caae de dca bc dfeee

(ae dcae).

Ie cdea ee aba aea a Bca de ee dfeecaee

ee.

{| ( ) |}i i i iMAD mediana M mediana M=

1

ii I

Iii

MADs

AD=

=


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O be dee de ae a defca de ee c dfeea fcaade ee ee ecd bc edad. E ed c d ecdbc, cacee e ad, ee, ee de ae bca defcaee e eea e eead de aea dfeeca ecd cacee ecd ad.

8.1.

O deeae de ae eee de de e eeead ee de de ea aa cada ee.Ea abdae ee e da ae ea defda:

Ma deeae ( ) Ma cae ( )

Maeacaee a de ea:[ ]

j jE Y X=

de:

jy c dad de ee aa ee;

X a a deeae; j e de cefcee.

8.1.1. D

Ea a eeea a dfeee aa de RNA e e bdada . a a de cefcee de cada a eeea e cada cacede a cefcee. O cefcee dee e deedee.Vea a be a deeae e Te.

E:

Cdee deeae c de caa da aa (A e B)deaee aa de a efeca c (Ref).

Qad a deeae c deee dca eed a d cefcee.

Cada e eeead faede a aa a a da feca e aaa a bae da feca. A 1 e eeead (A Ref), de 2e eeead (Ref A) (A Ref) e de 3 e eeead

(B Ref):


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1 A Ref

2 (A Ref)

3 B Ref

array

array

array

=

Se cdea c cefcee (A Ref) e (B Ref), ee, ee:

A a a deeae dee e a a X a e:

1Ref

2Ref

3

arrayA

array X B

array

=

A Ref Ref

(A Ref)Ref

B Ref

AX

B

=

L a a deeae e:

1 1 0

2 1 0

3 0 1

array

array

array

O a (1) a eda a dca e e e de cae ( ) 2.

Te e:

A Ref 1 0A Ref

(A Ref) 1 0 xB Ref

B Ref 0 1

=

E :

1

2

3

y 1 0A Ref

E y 1 0 xB Ref

y 0 1

=

Ref

Refj

A

B

=


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Obee e de aca caa aea A c Ref e B c Ref, acaa de eee e ea A c B fea. Paa caa a aa A eB dee de a a a cae ee ca de eee

eecfca ee cefcee e .

Fae e e j ced cefcee B A:

A Ref

B Aj

=

Tee aa:

A Ref Ref

(A Ref)A

B Ref

AX

B

=

A a a deeae dee e:

1 0

1 0

1 1

X

=

Te e:

A Ref 1 0 A Ref (A Ref) 1 0 x

B AB Ref 1 1

=

E :

1

2

3

y 1 0A Ref

E y 1 0 xB

y 1 1A

=

Obee e ee de c cefcee de eee (B A).De de aa de ea aa cada ee cefcee ead ed d adad e bed a ee de fcca.

1( ) . ( ' ) 'E Y X X X X Y = =


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8.1.2. C

C a a deeae d cefcee dee e deedee dece de a cae de eee ea cd. Deee e faea a a aa c ee cefcee a ae, a a cae.

Ea a ee e cefcee defd ea a deeae eacbad e cae de eee.Cada cae cede a caa de eee ee a aa de RNA.Paa eee e a a cae de e da.

O cae de eee dad :

T

j jC =

de C a a cae.

E:

Cdee deeae e c e aa:

O ead de e eee cae , eB A C B C A ,

, a a deeae defe aea d dee cefcee ee deee deedee. Eea de a a a cae aa faecbae d d cefcee defd ea a deeae e be cae de eee.Cdee de e e a a deeae defe caeB A e C B:

1 1 0

2 0 1

1 13

j

j

j

YB A

YE

C BY

=

Ode:

Ma deeae:

1 0

0 1

1 1

X

=

;

Ve de cefcee: jB A

C B

=

.


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Tad a a cae:

1 0 1

0 1 1C

=

Te e cefcee de eee bd T

j jC =

A:

0 1

1 0

1 1

j j

C BB A

B AC B

C A

= =

8.2. t

E ea e ed c e de a hn ee, e, a

a eaa da aca ee e c a de bedade e e e, Se e Ra (2004).Paa ca ee bea a e S (2004) a abdae Baeaaeca.

8.3. A B E

A eaca ada aa ae de fcca a eaca tdeada. Te aea eea e a eaca tda ece e e ade fadead a d ee, , eaad e de a a c,ad de Baea, S (2005).A eaca deada cd a ae d e d e a eaca da, c ece d a de bedade e aead, efed acfabdade acada c e ade aad.Oa eaca ada a eaca B lods ( log( )odds ), a da

babdade d ee e dfeecaee ee, S (2004).

8.4.

Cdee e aa cada eejf cdd ee de ee aa efcae dfeea de ee. Da e cc e ee j1 e j2 dfeeeee ee, c e de fcca dda 0,05= . Ifca e a babdade de fa de 5%, , de j1 edfeeeee ee e ea defcad ee c a. O e ae aaj2.Cdee aa a babdade de e ab ee ea defcad

ceaee: ad deedca ee a feca, e e ea


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babdade dada 2(1 0,05) 0,9025 = . eda e e a babdade

de ea ceed a e e de1 0, 9025 0, 0925 = . O ea, aea deea ead e de 5% ddaee, ad cdea ababdade de da a a afae eae cea c de eeeaad, ea dad c e ae da ee a, Se e Ra (2004).Qa a e de ee eeada, de ee defcad edee c, a e a babdade de e aa fa .Paa ca a aa de e ba de a d de Bfe ae FDR, ee, e aa e dec dda, aad ce da aa de faa decbea.

9.

9.1.

9.1.1. B

O eee cecd a eaa de .Ee eee f eaad ad ee c ade aa ed de cece e eebad. a a ee BMP2 e afea e da/ea d c daa.

:

O be d eee defca ee c ee aeada ee ca a e ea a ee d eae.

F 9.1:Pee Zeba ()

D:

4 e d ae de .


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F 9.2:Deeae d eee S

:

8.448 da;

4 4 (bc);

22 24 .

F 9.3:a d d eee S.

O bac de dad e de aa dad e:://bf.e.ed.a/aGUI/DaaSe.

9.1.2. B

O eee AA f ae de ed be eab de d ecebdade aecee e a Dd (2000).

A A (AA) ee ae eab d cee HDL.Ra c eeAA e e ba de cee HDL.

:

O be dee eee f caa ee c e de ee aeada

fad de a c a ce.

D:

Paa cada d 16 a cDNA acad f bd d RNA ac eeae d ad cae fecee ee C5. A aa efeca ada eda a bdae fa eaada ad cDNA d 8 a ce efa d c cae fecee ede C3, Dd (2001).


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F 9.4:A efeca c f fea ad RNA d a a, aaa aa fada c RNA de cada a, afa ce e a fa aae (a ).

Fa ad 16 a:

F 9.5:Deeae d eee AAI.

:

Cada microarray e 6.384 da de cDNA ea e aa 44 (16 bc),de cada bc e 19 a e 21 ca.

9.1.3.

Aaae d bac de dad ad fae eac e R e ace d Bcdc.


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B e de fae de cd e deee abe

a e feaea aa ae e cee de dad ec. Fca

abee caca eac e .

9.2.

Aaae dad ad a abdae de de eae, a a eeeada ace d Bcdc. Paa efca de eedfeeca ae a abdae Baeaa eca e, aa ca a aa defa ae c FDR.

10. B.

10.1. B

Icaee eca e aad R e Bcdc.

1

Faa dad e ae fae R. Ee e de aa dad e:

://ca.ec./b/d/bae/.

2

Iae ace d Bcdc. Baa da deaee R cadaba ( ece ce c a ee):

> source("http://www.bioconductor.org/getBioC.R")> getBioC()

Ob.: N de b ( >) e ca a a.

De de Iaad e Bcdc, de e aa a ae.

10.2. A

10.2.1. B

1


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Faa dad d Bac de Dad. E de aa dad a a:://bf.e.ed.a/aGUI/DaaSe..A dad eaa a.

2

Mda de aa a aa de fa ead a d Bac de Dad.

> setwd("C:/RBRAS_2009/Swirl")> getwd()[1] "C:/RBRAS_2009/Swirl"> dir()[1] "fish.gal" "SpotTypes.txt" "swirl.1.spot"[4] "swirl.2.spot" "swirl.3.spot" "swirl.4.spot"

[7] "SwirlSample.txt"

3

Caea ace :

> library(limma)

4

Caea be e a c fa fea a bdae (a ).

> targets targets

SlideNumber FileName Cy3 Cy5 Date1 81 swirl.1.spot swirl wild type 2001/9/202 82 swirl.2.spot wild type swirl 2001/9/203 93 swirl.3.spot swirl wild type 2001/11/84 94 swirl.4.spot wild type swirl 2001/11/8

5

Caea be c a edade de ee (e a c ee. e c e de ee d ee).C a ae dee eee fa aaada e fae , dadde e d e . Deee dca e ee dad eee dfae , ee a ea a dee fae.

> RG


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Read swirl.3.spotRead swirl.4.spot> names(RG)[1] "R" "G" "Rb" "Gb" "targets"

[6] "source" "genes" "printer"

Ca a ae ee d aaada e e faee caa de e a de dad, ee dea e aad e a de e a fa de a a, de fa e cada a eeee ee e cada ca a bda. A dee e dad deaee Rad cad:

> read.table()

cad

> read.delim()

6

Acecea a be RG a caa, e e de cada ee (ea faee a c ee .a).

> RG$genes names(RG)[1] "R" "G" "Rb" "Gb" "targets"[6] "source" "genes"> RG$genes[1:10,]

Block Row Column ID Name1 1 1 1 control geno12 1 1 2 control geno23 1 1 3 control geno34 1 1 4 control 3XSSC5 1 1 5 control 3XSSC6 1 1 6 control EST17 1 1 7 control geno18 1 1 8 control geno29 1 1 9 control geno310 1 1 10 control 3XSSC

7

C ea ad ada ., a de e 442224 ab

eca e fad. Fae ad a .a.


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> RG$printer RG$printer$ngrid.r[1] 4

$ngrid.c[1] 4

$nspot.r[1] 22

$nspot.c[1] 24

attr(,"class")[1] "PrintLayout"

8

Fae a ce d .O cad:

> RGb


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F 10.1: MA d a d eee .

10

Fae MA d de e aa cada aa.

> plotPrintTipLoess(MA, array=1)> plotPrintTipLoess(MA, array=2)> plotPrintTipLoess(MA, array=3)> plotPrintTipLoess(MA, array=4)


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F 10.2:Gfc MA d de e (bc), e aa, aa 1, 2, 3 e 4 eecaee d eee .

11

Naa de d :

> MA plotPrintTipLoess(MA, array=1)> plotPrintTipLoess(MA, array=2)> plotPrintTipLoess(MA, array=3)> plotPrintTipLoess(MA, array=4)


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F 10.3: Gfc MA d de e (bc), a a aade d , aa 1, 2, 3 e 4 eecaee d eee .

12

Fae b de cada .

> boxplot(MA$M~col(MA$M),names=colnames(MA$M),col=rainbow(4))


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F 10.4: B d a d eee .

P ee fc de bea e a edade e ceada a dae, ea, fa cda a ca ea aa de d ,eea a aa dfeee, e dee fae a aaee aa ce de ecaa.

13

Fae a aa de ecaa ee .

> MA boxplot(MA$M~col(MA$M),names=colnames(MA$M),col=rainbow(4))

F 10.5: B d a d eee de da aaee .


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14

Aae de ea aa cada ee.

> design fit names(fit)[1] "coefficients" "rank"[3] "assign" "qr"[5] "df.residual" "sigma"[7] "cov.coefficients" "stdev.unscaled"[9] "pivot" "genes"

[11] "Amean" "method"[13] "design"

15

Caca a eaca de ee ad a abdae baeaa eca.

> fit names(fit)[1] "coefficients" "rank"[3] "assign" "qr"[5] "df.residual" "sigma"[7] "cov.coefficients" "stdev.unscaled"

[9] "pivot" "genes"[11] "Amean" "method"[13] "design" "df.prior"[15] "s2.prior" "var.prior"[17] "proportion" "s2.post"[19] "t" "p.value"[21] "lods" "F"[23] "F.p.value"

16

Tabea de Gee Dfeecaee Ee

> options(digits=3)> topTable(fit,number=30,adjust="fdr")Block Row Column ID Name logFC AveExpr t P.Value adj.P.Val B

3721 8 2 1 control BMP2 -2.21 12.1 -21.1 1.03e-07 0.000357 7.961609 4 2 1 control BMP2 -2.30 13.1 -20.3 1.34e-07 0.000357 7.783723 8 2 3 control Dlx3 -2.18 13.3 -20.0 1.48e-07 0.000357 7.711611 4 2 3 control Dlx3 -2.18 13.5 -19.6 1.69e-07 0.000357 7.628295 16 16 15 fb94h06 20-L12 1.27 12.0 14.1 1.74e-06 0.002067 5.787036 14 8 4 fb40h07 7-D14 1.35 13.8 13.5 2.29e-06 0.002067 5.54

515 1 22 11 fc22a09 27-E17 1.27 13.2 13.4 2.44e-06 0.002067 5.485075 10 14 11 fb85f09 18-G18 1.28 14.4 13.4 2.46e-06 0.002067 5.487307 14 19 11 fc10h09 24-H18 1.20 13.4 13.2 2.67e-06 0.002067 5.40


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319 1 14 7 fb85a01 18-E1 -1.29 12.5 -13.1 2.91e-06 0.002067 5.322961 6 14 9 fb85d05 18-F10 -2.69 10.3 -13.0 3.04e-06 0.002067 5.294032 8 14 24 fb87d12 18-N24 1.27 14.2 12.8 3.28e-06 0.002067 5.226903 14 2 15 control Vox -1.26 13.4 -12.8 3.35e-06 0.002067 5.204546 9 14 10 fb85e07 18-G13 1.23 14.2 12.8 3.42e-06 0.002067 5.18683 2 7 11 fb37b09 6-E18 1.31 13.3 12.4 4.10e-06 0.002182 5.02

1697 4 5 17 fb26b10 3-I20 1.09 13.3 12.4 4.30e-06 0.002182 4.977491 15 5 3 fb24g06 3-D11 1.33 13.6 12.3 4.39e-06 0.002182 4.964188 8 21 12 fc18d12 26-F24 -1.25 12.1 -12.2 4.71e-06 0.002209 4.894380 9 7 12 fb37e11 6-G21 1.23 14.0 12.0 5.19e-06 0.002216 4.803726 8 2 6 control fli-1 -1.32 10.3 -11.9 5.40e-06 0.002216 4.762679 6 2 15 control Vox -1.25 13.4 -11.9 5.72e-06 0.002216 4.715931 12 6 3 fb32f06 5-C12 -1.10 13.0 -11.7 6.24e-06 0.002216 4.637602 15 9 18 fb50g12 9-L23 1.16 14.0 11.7 6.25e-06 0.002216 4.632151 5 2 15 control vent -1.40 12.7 -11.7 6.30e-06 0.002216 4.623790 8 4 22 fb23d08 2-N16 1.16 12.5 11.6 6.57e-06 0.002221 4.587542 15 7 6 fb36g12 6-D23 1.12 13.5 11.0 9.23e-06 0.003000 4.274263 9 2 15 control vent -1.41 12.7 -10.8 1.06e-05 0.003326 4.136375 13 2 15 control vent -1.37 12.5 -10.5 1.33e-05 0.004026 3.91

1146 3 4 18 fb22a12 2-I23 1.05 13.7 10.2 1.57e-05 0.004242 3.76157 1 7 13 fb38a01 6-I1 -1.82 10.8 -10.2 1.58e-05 0.004242 3.75

17

Fae fc .Ee fc fe c: 2(fdcae) VS 10(a).Va caea be ced 2(fdcae) e ae aa d ee (eecfcae e d ee ccad cad be =(MA$M) e Tabe, de (MA$M) e de a de M, ea,

e a de ee ).

> y names(y)[1] "Block" "Row" "Column" "ID"[5] "Name" "logFC" "AveExpr" "t"[9] "P.Value" "adj.P.Val" "B"

C 2(fdcae) e a ca 6 e ae aad e a ca 10ae ea ca aa ca fc.

> plot(y[,6],-log(y[,10],10),xlab="log2(Fold-Change)",ylab="-log10(P.Value)",main="Volcano plot",cex=0.1,pch=19)> abline(v=c(-1,1),col="blue")> abline(h=-log(0.05,10),col="red")


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F 10.6:Gfc d eee .

O aca da a eea eeea ee c a e e0,05, ea, ee e aeea ee dfeeca c e de cfaade 5%. O ee aca da a eea e eeda da a a cedee 2(fdcae)= 1 e dea da a a cedee 2(fdcae)= 1 ee e e eea a d e da ee e a aa e ea a.

10.2.2. B

1

Faa dad d Bac de Dad. E de aa dad a a:://bf.e.ed.a/aGUI/DaaSe..A dad eaa a.

2

Mda de aa a aa de fa ead a d Bac de Dad.

> setwd("C:/RBRAS_2009/ApoAI")> getwd()[1] "C:/RBRAS_2009/ApoAI"> dir()[1] "ApoAI.gal" "ApoAISpotTypes.txt"[3] "ApoAITargets.txt" "c1.spot"[5] "c2.spot" "c3.spot"[7] "c4.spot" "c5.spot"


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[9] "c6.spot" "c7.spot"[11] "c8.spot" "k1.spot"[13] "k2.spot" "k3.spot"[15] "k4.spot" "k5.spot"[17] "k6.spot" "k7.spot"

[19] "k8.spot"

3

Caea ace :

> library(limma)

4

Caea be e a c fa fea a bdae (a ).

> targets targets

SlideNumber Name FileName Cy3 Cy51 1 c1 c1.spot Ref wild type2 2 c2 c2.spot Ref wild type3 3 c3 c3.spot Ref wild type

4 4 c4 c4.spot Ref wild type5 5 c5 c5.spot Ref wild type6 6 c6 c6.spot Ref wild type7 7 c7 c7.spot Ref wild type8 8 c8 c8.spot Ref wild type9 9 k1 k1.spot Ref ApoAI KO10 10 k2 k2.spot Ref ApoAI KO11 11 k3 k3.spot Ref ApoAI KO12 12 k4 k4.spot Ref ApoAI KO13 13 k5 k5.spot Ref ApoAI KO14 14 k6 k6.spot Ref ApoAI KO

15 15 k7 k7.spot Ref ApoAI KO16 16 k8 k8.spot Ref ApoAI KO

5

Caea be c a edade de ee (e a c ee. e c e de ee d ee).C a ae dee eee fa aaada e fae , dadde e d deaee a. Deee dca e ee dad

eee d fae , ee a ea a dee fae.


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> RG names(RG)[1] "R" "G" "Rb" "Gb" "targets"[6] "source"

6

Acecea a be RG a caa, e e de cada ee (ea faee a c ee .a).

> RG$genes names(RG)[1] "R" "G" "Rb" "Gb" "targets"[6] "source" "genes"> RG$genes[1:10,]

Block Row Column ID Name CloneID1 1 1 1 Control Cy3RT BLANK2 1 1 2 Control Cy5RT BLANK3 1 1 3 cDNA mSRB1 mSRB1

4 1 1 4 BLANK BLANK BLANK5 1 1 5 BLANK BLANK BLANK6 1 1 6 BLANK BLANK BLANK7 1 1 7 cDNA 3174488 1 1 8 cDNA 5'similartoSW:BTF3_H 3174529 1 1 9 cDNA 5'similartogb:J04794 31745610 1 1 10 cDNA 5'.gi|1287586|gb|W13 317460


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7

C ea ad ada SPOT, a de e 441921 abeca e fad. Fae ad a GAL.

> RG$printer RG$printer$ngrid.r[1] 4

$ngrid.c[1] 4

$nspot.r[1] 19

$nspot.c[1] 21

attr(,"class")[1] "PrintLayout"

8

Fae a ce d .

> MA names(MA)[1] "targets" "source" "genes" "printer" "M"[6] "A"

9

Fae MA d :

> plotMA(MA, array=1)> plotMA(MA, array=2)

.

.

.> plotMA(MA, array=15)> plotMA(MA, array=16)


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..

....

F 10.7: MA aa aa 1, 2, 15 e 16, eecaee, aa eeeAA.

10

Fae Ad de e aa .

> plotPrintTipLoess(MA, array=1)> plotPrintTipLoess(MA, array=2)

.

.

.> plotPrintTipLoess(MA, array=15)> plotPrintTipLoess(MA, array=16)


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..

....

F 10.8:Gfc MA d de e (bc), e aa, aa 1, 2, 15 e 16 eecaee d eeeAA.

11

Naa de d :

> MA plotPrintTipLoess(MA, array=1)


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> plotPrintTipLoess(MA, array=2)...

> plotPrintTipLoess(MA, array=15)> plotPrintTipLoess(MA, array=16)

.

.

.

.

.

.

F 10.9: Gfc MA d de e (bc), a a aade d , aa 1, 2, 15 e 16 eecaee d eeeAA.

12

Fae b de cada .

> boxplot(MA$M~col(MA$M),names=colnames(MA$M),col=rainbow(16))


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F 10.10: B d 16 d eeeAA.

13

Fae a aa de ecaa ee .

> MA boxplot(MA$M~col(MA$M),names=colnames(MA$M),col=rainbow(16))

F 10.10: B d 16 d eeeAAa a aa ee .


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14

Aae de ea aa cada ee.

> design design

Control-Ref KO-Control[1,] 1 0[2,] 1 0[3,] 1 0[4,] 1 0[5,] 1 0[6,] 1 0[7,] 1 0[8,] 1 0

[9,] 1 1[10,] 1 1[11,] 1 1[12,] 1 1[13,] 1 1[14,] 1 1[15,] 1 1[16,] 1 1> fit names(fit)[1] "coefficients" "stdev.unscaled"

[3] "sigma" "df.residual"[5] "cov.coefficients" "pivot"[7] "genes" "Amean"[9] "method" "design"

15

Caca a eaca de ee ad a abdae baeaa eca.

> fit names(fit)[1] "coefficients" "stdev.unscaled"[3] "sigma" "df.residual"[5] "cov.coefficients" "pivot"[7] "genes" "Amean"[9] "method" "design"

[11] "df.prior" "s2.prior"[13] "var.prior" "proportion"[15] "s2.post" "t"

[17] "p.value" "lods"[19] "F" "F.p.value"


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16

Tabea de Gee Dfeecaee Ee

> names(topTable(fit,coef=2, number=30,adjust="fdr"))[1] "Block" "Row" "Column" "ID"[5] "Name" "CloneID" "logFC" "AveExpr"[9] "t" "P.Value" "adj.P.Val" "B"

> options(digits=3)> topTable(fit,coef=2, number=30,adjust="fdr")

Name logFC AveExpr t P.Value adj.P.Val B

2149 ApoAI,lipid-Img -2.930 12.46 -16.81 1.93e-12 1.23e-08 11.0025356 CATECHOLO-METHYLTRAN -1.678 12.92 -12.73 1.98e-10 6.32e-07 9.239540 EST,HighlysimilartoA -2.822 12.28 -12.25 3.70e-10 7.88e-07 8.9564139 EST,WeaklysimilartoC -0.883 12.60 -10.16 7.04e-09 1.12e-05 7.4761739 ApoCIII,lipid-Img -0.828 13.74 -8.83 5.87e-08 7.49e-05 6.2581496 est -0.866 12.23 -7.92 2.84e-07 3.03e-04 5.2732537 ESTs,Highlysimilarto -0.911 13.63 -7.77 3.72e-07 3.39e-04 5.0994941 similartoyeaststerol -0.793 13.29 -5.91 1.35e-05 1.08e-02 2.602947 EST,WeaklysimilartoF -0.504 10.54 -4.46 3.03e-04 2.15e-01 0.2313920 ESTs,Weaklysimilarto 0.345 10.78 3.95 9.41e-04 5.59e-01 -0.6676073 estrogenrec 0.416 9.78 3.93 9.86e-04 5.59e-01 -0.7055693 Meox2 0.517 9.77 3.90 1.05e-03 5.59e-01 -0.755

1347 Musmusculustranscrip -0.376 10.18 -3.73 1.52e-03 6.58e-01 -1.0523510 MousemRNAfortypeIIDN 0.366 10.31 3.69 1.69e-03 6.58e-01 -1.135654 BLANK -0.447 9.37 -3.67 1.75e-03 6.58e-01 -1.1644140 APXL2,5q-Img -0.377 9.78 -3.65 1.82e-03 6.58e-01 -1.1952009 5'.gi|1285734|gb|W11 0.426 9.63 3.65 1.83e-03 6.58e-01 -1.202954 Caspase7,heart-Img -0.276 12.15 -3.63 1.93e-03 6.58e-01 -1.2445692 Olf-1 0.441 9.77 3.62 1.96e-03 6.58e-01 -1.2555986 Cy3RT 0.870 10.24 3.59 2.10e-03 6.70e-01 -1.3101337 psoriasis-associated -0.717 11.67 -3.56 2.24e-03 6.80e-01 -1.3624943 ADENOSINEA1RECEPTOR, -0.371 11.73 -3.51 2.48e-03 7.03e-01 -1.4455562 BLANK 0.396 8.66 3.50 2.53e-03 7.03e-01 -1.4624892 FGF12A 0.431 9.74 3.46 2.78e-03 7.32e-01 -1.537

634 MDB1376 -0.334 9.31 -3.37 3.44e-03 7.32e-01 -1.7091224 5'.gi|1287131|gb|W12 0.512 10.18 3.32 3.78e-03 7.32e-01 -1.7855343 NCAM-120,Brain-Img -0.392 10.75 -3.30 3.99e-03 7.32e-01 -1.8281972 BLANK -0.331 8.43 -3.28 4.16e-03 7.32e-01 -1.8622352 ESTs,Moderatelysimil -0.405 9.14 -3.28 4.18e-03 7.32e-01 -1.865834 0.300 10.25 3.27 4.28e-03 7.32e-01 -1.884

17

Fae fc .

> y


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> names(y)[1] "Block" "Row" "Column" "ID"[5] "Name" "CloneID" "logFC" "AveExpr"[9] "t" "P.Value" "adj.P.Val" "B"

> plot(y[,7],-log(y[,10],10),xlab="log2(Fold-Change)",ylab="-log10(P.Value)",main="Volcano plot",cex=0.2,pch=19)

F 10.11: Gfc d eeeAA.

10.2.3. A

.

Pde aaa eee de a c c e fe eee de dace c efeca c.

Se e, ee, a c,cada d fad d A eB, dea ca c deeae c efeeca c ada a deeae:

1 0

1 0

1 1

1 1

X

=

e eecfca cae:


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A-Ref

B - Aj

=

Obee e a efeeca c ee, , cad topTable

fa e ee ead efeee a cefcee 2(topTable(fit,coef=2)), e caa A e B.

A def a a deeae aa de ea c cadlmFit() e caca a eaca de ee e ae c cadeBayes().

11. B

CARAZZOLLE, M. F., (2008) Ae de caa de DNA. De e:://.e.b.ca.b/eeea2008/e/caa.df.

DUDOIT, S. e a. Saca ed f def deea eeed ee ecaed cDNA caa eee. 2000.

DUDOIT, S. e a. Naa f cDNA Mcaa Daa. 2001.

EISEN, M. ScaAe Ue Maa. Safd Ue; 1998. De e:://aa.b./EeSfae..

ESTEVES, G.H. Vada de cede aa edda de ee ca a a

de ae de cDNA . S Pa; 2002. [Dea de Mead

Fda A Pdee].

ESTEVES, G.H. Md eac aa a ae de dad de cDNA caa e

abee caca ead. S Pa; 2007. [Tee de Dad

Uedade de S Pa].

FUJITA, A. Ae de dad de ee ca: aa de e

deae de ede eaa. S Pa; 2007. [Tee de Dad

Uedade de S Pa].

HADDAD, S. R., APLICAO DE MODELOS LINEARES PARA ANLISE DE EXPRESSOGNICA EM EXPERIMENTOS DE MICROARRAYS. Bca; 2007. [Dea deead UNIVERSIDADE ESTADUAL PAULISTA FACULDADE DE MEDICINAVETERINRIA E ZOOTECNIA].

LOPES, F. C.; PAIS, H. L. M., (J. 2006) Rea de Pec de Fa de C.


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Uedade de Ae, Ae, Pa.

MARQUES, E. K. Dac Gec Meca. 1. ed. Caa: Ed. ULBRA, 2003. 372

.

PEREIRA, R. N., Ce d e d I e eee de ceca. Laa; 2008. [Dea de ead Uedade Fedea de Laa].

SCHENA, M. e a., (J. 1998) Mcaa: bec' dce af f

fca ec BC 16:301306.

SMYTH, G. K. Lea de ad eca bae ed f ae dffeeaee caa eee. Saca Aca Geec ad

Meca B, . Ace 3, 2004.

SMYTH, G. K. (2005). La: ea de f caa daa. I: B C B B, R. Geea, V. Cae, S.Dd, R. Ia, W. Hbe (ed.), Se, Ne Y, ae 397420. (Pbe ebe, PDF).

SOLER, J. P., ROSA, F. H. F. P. (2005). Rea be Pe Ua de deac e deee de feaea e bfca aa ae decDNA ad caa. Pe FAPESP 03/020740. S Pa.

De e: ://.fefea.e/fe/a/eafae_20041210.df.

SOUTO, M. C. P., Ee Gca. De e

://.da.f.b/ac/BIOINFORMATICA/BIO2004.1/BIOaa11

eeaeca..

STAFFORD, P. (2008). Med Mcaa Naa.

THORNE, N., Udead e de a ea de f caaeee. De e:

://.da.ca.ac./e/22/22ebae/deMaLec.df.

Ya, Y. H., Dd, S., L, P., L, D. M., Pe, V., Na, J., ad Seed, T. P. (2002).Naa f cDNA caa daa: a b ce ed adde ead e de eac aa. A 30(4):e15.

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