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Berkeley STATISTICS 246 - Introduction to microarray technology Extra material

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1Introduction to microarray technologyExtra materialLecture 17, Statistics 246March 18, 20042Back to cDNA arrays: the M-GuideBuild your own arrayerM-GuideArray Maker DocumentationPrinting Microarrays3Printing MicroarraysPrint HeadPlate HandlingXYZ positioningRepeatability & AccuracyResolutionEnvironmental ControlHumidityDustInstrument ControlSample Tracking Software4Ngai Lab arrayer , UC Berkeley5Microarray Gridder6Slide Preparation: Home GrownProtocol for preparing poly-L-Lysine slides forMicroarrays1. Wash 180 slides completely2. Prepare poly-lysine solution3. Pour solution over slide4. Rinse, spin dry and store slides5. Use slides no less than 2 and no morethan 4-6 months later78Protocol for Amplifying Products to Print on ArrayAll PCR reactions in 96-well format, 100 ml reaction volumePerform PCR reactions in a Tetrad MachineReactions are assayed on 96 well agarose gelNeed multi-channel pipetting systemAlso desirable to have Multimek 96-well pipetting robotProduct Amplification and preparation: What to Print?9MJ Tetrad PCR machine10Protocol for preparation of Plasmid DNA fromBacterial Clones Containing Mammalian DNA1. Inoculate a deep 96-well plate filled with IB (+ antibioticmarker) with a small amount of bacterial culture. Incubatewith shaking at 37˚C2. Spin down the cultures and follow the manufacturer’sprotocol for the QIAprep3. Use 1-5 ul of eluted plasmid DNA as PCR template11Protocol for precipitation and 384 WellArraying of PCR products1. After running reactions on 1% agarose gel and documenting results,add sodium acetate, pH 5.5 and 110 ul room temp isopropanol2. Transfer reactions to U-bottom plates,.. tape plates together.3. Spin plates at 4.500 rpm for 2 hours4. Carefully aspirate solution5. Add 100ul 70% EtOH. Spin plates for another hour at 4,5006. Aspirate again and let air dry or dry in a 96 well speed-vac7. Allow PCR products to resuspend in 20ul of H2O for at least 18 hours8. Transfer products to 384 -well printing plates9. Dry plates down in speed-vac and resuspend products in 3X SSC10. Let plates resuspend overnight before printing.1213Printing ApproachesNon - ContactPiezoelectric dispenserSyringe-solenoid ink-jet dispenserContact (using rigid pin tools, similar to filter array)TweezerSplit pinMicro spotting pin14Micro SpottingpinMicro Spottingpin1516Practical ProblemsSurface chemistry: uneven surface may lead to highbackground.Dipping the pin into large volume -> pre-printing to drain offexcess sample.Spot variation can be due to mechanical difference betweenpins. Pins could be clogged during the printing process.Spot size and density depends on surface and solutionproperties.Pins need good washing between samples to preventsample carryover.17Post Processing ArraysProtocol for Post Processing MicroarraysHydration/Heat Fixing1. Pick out about 20-30 slides to be processed.2. Determine the correct orientation of slide, and if necessary,etch label on lower left corner of array side3. On back of slide, etch two lines above and below center ofarray to designate array area after processing4. Pour 100 ml 1X SSC into hydration tray and warm on slidewarmer at medium setting5. Set slide array side down and observe spots until properhydration is achieved.6. Upon reaching proper hydration, immediately snap dry slide7. Place slides in rack.18Surface blocking1. Store succinic anhydride in vacuum dessicator until ready foruse.2. Measure 335 ml 1-methly-2-pyrrolidinone into designated cleandry slide dish with stir bar3. Dissolve 5.5 g succinic anhydride completely4. IMMEDIATELY after succinic anhydride dissolves, mix in 15 ml1M NaBorate pH 8.0 and submerge slides in solution. Shakeevenly under level of solution.5.Soak slides in solution on shaker for 15’6. Before 15’ incubation is done, reduce heat on boiling water sotemp is approx 95C and no more bubbles are present. Drainexcess blocking solution off slides and transfer slide rack toboiling water and incubate for 1’30”7. Transfer rack to dish of 95% EtOH and plunge 5X. Spin down ontabletop.8. Arrays may be used immediately or stored for future use.19Isolating Nucleic Acid: “RNA, Membranes, and Tumors”Protocol for Total RNA isolation in S. CerevisaeModified FastTrack Protocol for Yeast Poly-A RNA IsolationProtocol for Poly-A IsolationsRevised Protocol for FastTrack mRNA extraction fromHuman CellsTumor mRNA isolationGradient-based membrane-bound Polysome ProtocolProtocol for Immunoprecipitation of Chromatin from FixedYeast Beadbeater Method2021Protocol for Total RNA Isolation in S. Cerevisae1. Spin down cells (about 250ml at OD600=0.5). Dump supernatant.2. Resuspend in 12 ml of AE Buffer. Transfer to Oak Ridge phenolresistant centrifuge tubes.3. Add 800 ul 25% SDS, 12 ml acid phenol. Mix well.4. Incubate 10’ at 65 ˚C, vortexing every minute.5. Incubate 5’ on ice.6. Spin down 15 minutes at 10,000 rpm in SS34 rotor7. Dump supernatant into pre-spun 50 ml PhaseLock tube.Add 15 mlchloroform and shake to mix (…ctd)228. Spin down 10’ at 3,000 rpm in table-top centrifuge9. Dump supernatant into new oak Ridge tube10. Add 1/10 volume 3M NaAcetate and equal volumeof room temperature isopropanol11. Spin down 35’-40’ at 12,000 rpm in SS3412. Wash with 70% EtOH, resuspending pellet, spinagain 20’ at 12,000 rpm13. Dump off EtOH. Dry pellet in vacuum oven briefly14. Resuspend in 500ul water15. Quantitate via spec and run 1ug on 1% agarose gel16. Store total RNA in -80˚CProtocol for Poly-A Isolations more complex: 18 steps.23Labelling Nucleic AcidProtocol for Reverse transcription and Amino-allyl Coupling ofRNAPreparation of Fluorescent cDNA Probe from Human mRNA(alternate protocol)Modified Eberwine (“ANTISENSE”) RNA AmplificationProtocolProtocol for labeling Genomic DNA for Microarrays: Version 1Genomic DNA Labeling ProtocolRound A/B DNA Ampification Protocol24Preparation of Fluorescent cDNA Probefrom Human mRNA (alternate protocol)1. To anneal primer, mix 2 ug of mRNA with 2 ug of a regular or anchored(5’-TTT TTT TTT TTT TTT TTT TT VN-3’) oligo-dT primer in a totalvolume of 15 ul (x 2)2. Heat to 70 ˚C for 10 min and cool on ice3. Add 15 ul of reaction mixture each to Cy3 and Cy5 reactions (5X firststrand buffer, 0.1M DTT, unlabeled dNTPs, Cy3 or Cy5, Superscript II4. 5X first strand buffer: 250 mM Tris-HCl, 375 KCl, 15mM MgCl25. Incubate at 42 ˚C for 1.5-2 hrs6. Degrade RNA by addition of 15ul of 0.1N NaOH, and incubate at 70 ˚C……(ctd)257. Neutralize by addition of 15 ul of 0.1N HCl,


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Berkeley STATISTICS 246 - Introduction to microarray technology Extra material

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