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AgnieszkaSzymczyk,EwaForma
115
RealTimePCR
InordertoanalyzeαKlothoexpression,totalRNAfrombladdercancercell
linewasisolatedusingTRIReagent(Ambion)followingthemanufacturer’s
protocol.cDNAwasobtained2µgRNAbyreversetranscriptionreactionusing
aHighCapacitycDNAReverseTranscriptionKit(AppliedBiosystems),
followingthemanufacturer’sprotocol.Real-timegeneexpressionanalysis
ofαKLgenewasperformedusingTaqMan®GeneExpressionAssaysconsisting
ofapairofunlabeledPCRprimersanTaqManprobeaccording
tomanufacturer’sinstruction.TheHPRT-1genewasusedasareferencegene.
TheassaynumbersforαKLandHPRT-1geneswasHs00183100_m1and
Hs02800695_m1(AppliedBiosystems)respectively.TheRealTimePCR
reactionwascarriedoutusingMastercycler®eprealplex(Eppendorf).
ThedifferencesinαKLexpressionwereanalyzedusingthe
∆
CTmethod
inreferencetoHPRT-1.
QuantitativeMSP-PCR
InordertoanalyzethemethylationstatusoftheCpGislandsinαKlotho
promoterregion,quantitativemethylation-specificPCR(MSP)analysis
wasperformed.DNAwasisolatedfromcelllinesusingTRIReagent(Ambion)
followingthemanufacturer’sprotocol.BisulfitemodificationofDNA
wasperformedusingtheEZDNAMethylationGoldKit(ZymoResearch,CA)
accordingtothemanufacturer’srecommendations.Bisulfite-convertedgenomic
DNA
was
amplified
using
specific
MSP
primer
for
unmethylated
andmethylated
promoter
region
of
αKlotho:
forward
unmethylated
GTGTTTGTTGGAGTGGCTGT;
reverse
unmethylated
CACCATAAA-
CATCCTACAAACAC;forwardmethylatedCGTTTGTTGGAGCGGTTGC;
reversemethylatedGCCATAAACGTCCTACAAACG.TheFastSYBRGreen
MasterMix(KAPPA)wasused.ThentheCTofthemethylatedandthe
unmethylatedproductswasusedtocalculatethepercentageofmethylationfor
αKlothogene.
