Chemistry Reference
In-Depth Information
RNA isolation
Sample A
Sample B
cDNA preparation
cDNA fluorescence labeling
Hybridization of cDNA to the array
Spotted
DNA
fragments
Figure 12.4
Microarrays used to study gene expression. mRNA is converted to complementary DNA (cDNA)
and tagged with a luorescent label following sample extraction. cDNA luorescent samples are
then applied to a microarray that contains DNA fragments corresponding to thousands of genes.
Fluorescence intensity in each spot is used to estimate levels of gene expression. (Adapted from
Varzakas, T. et al., in
Tomatoes and Tomato Products, Nutritional, Medicinal and Therapeutic
Properties
, edited by V.R. Preedy and R.R. Watson, pp. 515-536, Science Publishers, Enield,
NH, 2008. With permission.)
species.Toensurethereliabilityofthismethod,differentkindsofpositiveandnegativecontrolsare
usedintheDNAmicroarray.
12.4.1.5 Random Ampliication of Polymorphic DNA
RandomampliicationofpolymorphicDNA(RAPD)isatypeofPCRreactionwithDNAsequences
ampliiedrandomly.RAPDisappliedbyusingseveralarbitrary,shortprimers(8-12nucleotides)tothe
DNAbasedonthefactthatsomepartsoftheDNAwillamplify.Byresolvingtheresultingpatterns
byagarosegelelectrophoresis,itispossibletodifferentiatestrainsofthesameorganism.Themethod
hasbeenusedtoassesspopulationsofmRNAwithdifferentialdisplay(LiangandPaedee1992)or
arbitrarilyprimedPCR(Welshetal.1992),ampliicationofrandommRNAsubsets,andsubsequent
analysisoftheresultingfragmentpoolbygelelectrophoresis(Argyropoulosetal.2008).
12.4.1.6 Real-Time Polymerase Chain Reaction
Inreal-timePCR,thequantiicationofmRNAsequencesisaccomplishedbyabsoluteorrela-
tiveanalysismethods.Increasingly,therelativemethodofanalysisisbeingused,astrendsingene
expressioncanbebetterexplained,butresultsdependonreferencegenesnecessarytonormalize
samplevariations(McMaughandLyon2003;WeihongandSaint2002).
Acommontechniqueinrelativequantiicationisthechoiceofanendogenouscontroltonormal-
izeexperimentalvariationscausedbydifferencesintheamountoftheRNAaddedinthereverse
transcription(RT)PCRreactions.Speciicationsofreliableendogenouscontrols(i.e.,housekeeping
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