(thisprotocolwasadaptedbyJoeDeRisifromonedevelopedatRosettaInpharmatics)ResUSPendthecontentsofonepackofthemonofunctionalNHS-esterCy3orCy5dye(mono-functionalCy3orCy5reactivepack,AmershamcatalogPA23001andPA25001)in72ulDMSO.Aliquot16x4.5ulandeitheruseimmediatelyordrythealiquotsinaspeedvac.Storealiquotsat4Cinadessicator.UseonealiquotofthedyesforeachRTlabellingreactionandmicroarrayhybridization.
I.RTReaction
OligodT/RandomPrimeRNA:
| Concentration | uL |
OligodT:pdN6 | 5ug/uL | 1 |
TotalVol.ofRNA | | 14.5 |
IncubateRNA(1-4microgrampolyA+RNA)andoligodTat70Cfor10min.Chillonice10min.1xdNTPS:500uMeachdATP,dCTP,dGTP,200uMaadUTP,300uMdTTP | 50xrecipe:FOR2:310uLeach100mMdATP,dGTP,dCTP,4uL100mMaa-dUTP*,6uL100mMdTTP |
*5-(3-Aminoallyl)-2"-deoxyuridine5"-triphosphatesodiumsalt,Sigma-AldrichCatalogA-0410CDNAsynthesis
| Concentration | uL | 10.5 |
5xbuffer | | 6 | 63 |
50xaadUTP/dNTPs | | 0.6 | 6.3 |
DTT | 0.1M | 3 | 31.5 |
SuperScriptII | | 1.9 | 19.95 |
Water | | 3 | 31.5 |
| | 14.5 | 14.5 |
42degreesfor2hours | | Aliquots |
II.Hydrolysis
Add:10ul1NNaOH
10ul.5MEDTA
Incubate:15min.at65°C.
Neutralize:25ul1MTrispH7.4or1MHEPESpH7.5
III.CleanupTocontinuewiththeamino-allyldyecouplingprocedureallTrismustberemovedfromthereactiontopreventthemonofunctionalNHS-esterCyedyesfromcouplingtofreeaminegroupsinsolution.
FilloneMicrocon30concentratorwith450ulwater.
Addneutralizedreaction.
Spinat12Kfor8minutes.
Dumpflo-thru.
Repeatprocess2X,refillingoriginalfilter.
Elute.
Dryeluateinspeedvac.
IV.Coupling
ResuspendcDNApelletin9ul0.1MNABIcarbonateBufferpH9.0.AddthistothedriedaliquotofCy3orCy5dye.MixdyeandcDNA.
Letincubate1houratRTindark.
V.QuenchingandCleanupBeforecombiningCy3andCy5samplesforhybridizations,thereactionsmustbequenchedtopreventcross-coupling.
Add4.5ul4Mhydroxylamine.
Letreactionincubate15min.atRTindark.
Toremoveunicorporated/quenchedcyedyesproceedwithQia-quickPCRpurificationkit.
CombineCy3andCy5reactions.
Add70ulwater.Add500ulBufferPB.
ApplytoQia-quickcolumnandspinat13,000rpminfor30-60sec.
Aspirateoffflo-thru.
Add750ulBufferPEandspin30-60sec.
Aspirateoffflo-thruandrepeat.Aspirateflo-thruandspinfor1min.athighspeedtodrycolumn.
Transfertofreshepp.tube
Add30ulBufferEBtocenteroffilterandletsit1min.atRT.
Spinat13,000rpmfor1min.
Repeatelutionstepagain.
VI.Hyb.Prep.
DrydownQia-quickeluateinspeedvac.
Bringvolumeto18ulwithwaterandHEPESpH7.5sothatthefinalconcentration(afterSSCandotheradditions)is25mM.
Add:3.6ul20XSSC
1.8ulpolyA(10mg/ml)and/orothercompetitornucleicacid
Optional:filterinMillipore0.45micronspincolumn.Add:0.54ul10%SDS.
Incubatereactionat100°Cfor2min.
Applytopreparedmicroarray.