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Techniquestomeasurechangesin

geneexpression

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Severaltechniquesarecurrentlyavailableto

measurechanges

in

gene

expression.

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Becauseofitshighsensitivity,reverse

transcriptionwith

following

polymerase

chainreaction(RTPCR)isbeingincreasingly

usedtoquantifyphysiologicallyrelevant

changesin

gene

expression

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differentRT

PCR

quantification

methods

are

described

and

compared:

1externalstandardised RTPCRwithquantificationonethidium

bromidestained

gels

in

combination

with

densitometry;

2external standardised RTPCRwith onlinedetection usingLightCycler CybrGentechnology.

SybrGreen binds to

newly synthesised DNA

giving a

fluorescencesignal once percycle that isproportional totheDNA

concentration;

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RTPCRwithquantificationon

ethidium bromidestained

gels

in

combinationwithdensitometry

padresamostras

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RTPCRwithquantificationon

ethidium bromidestained

gels

in

combinationwithdensitometry

QuantificationofRTPCRproductsinethidium bromidestainedgelscouldbeperformedusingexternalcRNAstandards.

Withinalimited

range

of

linear

detection

(4

40pg

HAScRNA;

r=

0.984)a5folddifferencebetweentwodistinctcDNA samplescouldbedetected(fig.1).

Whencomparing

to

aLightCycler quantification

an

increase

of

the

reliabilityandsensitivitywasobserved(11000pgHAScRNA;r=0.996)tolatertechnique(fig.2).0

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LightCycler onlinequantification

padres

amostras

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Questes Numaexperinciadequantificao

deexpressogenticaporRTPCRemtemporeal,forampreparadosumasrie

de

padres

com

quantidades

respectivamente:

12pg

220pg

3200pg

a)Indique,nogrfico,acurvarespectivadefluorescnciaparacadapadro.

b)Indique,

no

grfico

as

curvas

correspondentesrespectivamenteamostraquantificadascom6pg ecom18pg

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Quantificaoabsoluta

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QuantificaorelativaMtodocomparativo

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Diminuio

relativadeCt s

48hemrelao

a0h

Aumentoua

quantidade

relativadeDNA

s48h!

Sobreexpresso

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Regulationofgeneexpressionineukaryoticcells

Themodelusedisamurineerythroleukemia cellline(MELcells).

Thesecontinuouslycycling,immatureredbloodcells,arrestedatanearlystagein

erythropoiesis,canbeinducedtoprogressfurtherthroughtheprocessby72h

exposureto1.8%DMSO.

ThisgivesacontrolcellsampleandaDMSOtreatedpreparation.

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Threesequenceswereselectedtocomparebetweenthecontrol(noDMSO)andtreated

cells(DMSO).

TwoweresignificantlyupregulatedwithDMSOtreatment:bglobin(bHb)and

aminolevulinate synthase(ALAS),

while

one

was

down

regulated:

Carbonic Anhydrase 1

(CA1).

Thesesequencesshowedlarge reproducible changes (>50fold)ingeneexpression

andcouldbelinkedtotheprocessoferythroid differentiationand function.

Genesalvo

analisarasalteraesdeexpressocomtratamentoporDMSO

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Inmanycasesthelevelofaparticularsequenceisexpressedrelativetoa

referencegene.

18SrRNA waschoseninthisexperimentasthereferencegeneasitdoesnot

changewith

DMSO

treatment

Genedereferncia controloendgeno

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Questes

Daobservao

dos

grficos

anteriores,

eSEM

efectuar

uma

anlise

quantitativa:

1 AquecorrespondemosdoisvaloresdeCt lidosnogrficoA?

2AquecorrespondemosquatrovaloresdeCt lidosnogrficoB?

3 Podeconfirmarsequeogene18SRNAfoibemseleccionadocomogenede

referncia?Porqu?

4 Podeestimarse(aproximaonoquantitativamente)qualdosgenessofrem

aumentooudiminuiodeexpressocomotratamento?Porqu?

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Assinaleos

valores

utilizadospara

calcularaproporo

deaumentode

expressodogene

Hb emclulastratadascomDMSO

Notratado

Tratado

==

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ToensurethatequivalentamountsofcDNAarebeingcomparedbetweencontroland

treatedsamplesthetargetgeneCtvaluesarenormalizedagainstthereferencegene,18S.

normalized Ct = Ct(target

sequence)

Ct

(reference

gene

18S)

Fig.3,

Column

H.

Oncethiscorrectionhasbeenperformed,thenormalizedCtvalues forthe

treatedaresubtractedfromtherespectivecontrol Ct values .

Byraisingthisdifference tothepowerof2,anestimateofthefoldchangebetween

controlandtreatedcanbeobtained.ThefoldchangesareshowninFigure3,ColumnsJ

(Treated:Control)andK(Control:Treated).

Theformulaisasfollows:

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ConsiderandoumgenealvoA

NaamostranotratadaadiferenaentreCt dogenealvoeCt do18S4.

NaamostratratadacomDMSO:

Ct genealvo=11

Ct de18S

=9

VerificouseexpressodiminudaouaumentadadogeneAapsotratamentocomDMSO?

Qualofactor

da

alterao

(quantas

vezes

alterada)?

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Graphicalpresentationsoftheresults.(A)

showstheresultsexpressedasfoldchange

(treated:control)foreach targetsequence

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Outrasquestes

Qualomodelo

biolgico

em

que

foram

analisadas

alteraes

da

expresso

gentica?

Emqueconsistiuotratamentodasclulas?

Quealteraesoreferidoqumicoinduznasclulas?

Quegenesalvoforamseleccionadosparaavaliaodealteraesdaexpresso?Porqu?

Quegenederefernciafoiseleccionado?Porqu?