1995- Mike Romanos- Advances in the use of Pichia pastoris for high-level gene expression.pdf
Exercícios - Techniques to measure changes in gene expression.pdf
Transcript of Exercícios - Techniques to measure changes in gene expression.pdf
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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?