Microarray

Basic introduction of DNA microarray

1. DNA
Microarray:
Sunil Bhandari
BalkumariCollege
©[email protected]

2. Microarray
Microarraysareusedinvariousareassuchas:
•Functionalgenomicsresearch(retrievingdifferentiallyexpressedgenes)
•Targetdiscovery(organismidentification,geneidentification)
•Biomarkerdetermination
•Pharmacology
•Toxicology(tofindeffectsofdrugsandtoxiccompoundsrespectivelyon
livingcells)
•Predictingdiseaseprognosis,and
•subclassifyingdisease
Microarraysareglassslidesthatcontain
particularmolecules(DNA-probes)attachedtotheir
surfacebyroboticarms,eachofwhichcan
specificallybind(hybridize)aparticulartarget
moleculeonoursample.
©[email protected]

3. Ingeneral,DNAmicroarraysare
microscopeslidesthatareprinted
withthousandsoftinyspotsin
definedpositions,witheachspot
containingaknownDNAsequence
orgenecalledProbe.
Average size of array in slide =1.28cm X
1.28cm = 1.6384cm2
Average size of each tiny spots =
0.005cm X 0.005cm = 0.000025cm2
Thus average number of spots in
microarray = 1.6384/0.000025 = 65,536
(suchtinyspotsmayrangesfrom65thousandto
6.5millionsperarray)
•Theprobesaredesignedwithaverage
of25basepairslong. ©[email protected]

4. Types of microarrays
©[email protected]

5. Process of microarray:
Sample
mRNA isolation
cDNA preparation
cDNA labelling
Mixing-hybridization
Washing Scan the array Array image
Image processing
Intensity data retrieval
Data expression
Fig: Process of Microarray
©[email protected]

6. Process of microarray:
1.mRNAmoleculesaretypicallyisolatedfrombothan
experimentalsampleandareferencesample.(Forexample,thereference
samplecouldbecollectedfromahealthyindividual,andtheexperimentalsamplecouldbe
collectedfromanindividualwithadiseaselikecancer.)
2.ThetwomRNAsamplesarethenconvertedinto
complementaryDNA(cDNA),andeachsampleislabeledwitha
fluorescentprobeofadifferentcolor.(Theexperimentalcdnasamplemay
belabeledwitharedfluorescentdye,whereasthereferencecdnamaybelabeledwitha
greenfluorescentdye.)
3.Thetwosamplesarethenmixedtogetherandallowedto
bindtothemicroarrayslide.TheprocessinwhichthecDNA
moleculesbindtotheDNAprobesontheslideiscalled
hybridization.
4.Followinghybridization,themicroarrayisscannedtomeasure
theexpressionofeachgenehybridizedontheslide.©[email protected]

7. 5.Iftheexpressionofaparticulargeneishigherintheexperimental
samplethaninthereferencesample,thenthecorrespondingspot
onthemicroarrayappearsred.Incontrast,iftheexpressioninthe
experimentalsampleislowerthaninthereferencesample,then
thespotappearsgreen.
7.Finally,ifthere
is equal
expressioninthe
twosamples,then
thespotappears
yellow.
The data gathered through microarrays can be used to create gene
expression profiles, which show simultaneous changes in the expression of
many genes in response to a particular condition or treatment.©[email protected]

8. Processing of image spots
©[email protected]

9. Expression of microarray data
1.Ratiodata
Ratiodataiscalculatedforcomparisonoftwoormoresample
basedontheamounttheirfluorescenceintensityproducedon
specificspots.Differentcolordye(Cy-3andCy-5)areusedtolabel
theindividualsampleandrespectivecolorproducedafter
illuminationiscaptured.Thesedatagivestheindividualaswellas
relativeinformationofthesampleunderstudy,
2.Intensitydata
Intensitydatarepresentsthemicroarrayoutcomesofindividual
dataofsingletypeofsamplelabelledwithsingletypeofdyes.Color
intensityoffluorescencegivestheinformationaboutidentification
ofsampleaswellasstrength/amountoftargetsinsample.Dataare
notmeantforcomparisonpurpose.
©[email protected]

10. Application of Microarray
©[email protected]