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Introduction to microarray technology Extra material

Introduction to microarray technology Extra material. Lecture 17, Statistics 246 March 18, 2004. Back to cDNA arrays: the M-Guide Build your own arrayer. M-Guide Array Maker Documentation Printing Microarrays. Printing Microarrays. Print Head Plate Handling XYZ positioning

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Introduction to microarray technology Extra material

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  1. Introduction to microarray technologyExtra material Lecture 17, Statistics 246 March 18, 2004

  2. Back to cDNA arrays: the M-Guide Build your own arrayer • M-Guide • Array Maker Documentation • Printing Microarrays

  3. Printing Microarrays • Print Head • Plate Handling • XYZ positioning • Repeatability & Accuracy • Resolution • Environmental Control • Humidity • Dust • Instrument Control • Sample Tracking Software

  4. Ngai Lab arrayer , UC Berkeley

  5. Microarray Gridder

  6. Slide Preparation: Home Grown • Protocol for preparing poly-L-Lysine slides for Microarrays • 1. Wash 180 slides completely • 2. Prepare poly-lysine solution • 3. Pour solution over slide • 4. Rinse, spin dry and store slides • 5. Use slides no less than 2 and no more than 4-6 months later

  7. Product Amplification and preparation: What to Print? Protocol for Amplifying Products to Print on Array • All PCR reactions in 96-well format, 100 ml reaction volume • Perform PCR reactions in a Tetrad Machine • Reactions are assayed on 96 well agarose gel • Need multi-channel pipetting system • Also desirable to have Multimek 96-well pipetting robot

  8. MJ Tetrad PCR machine

  9. Protocol for preparation of Plasmid DNA from Bacterial Clones Containing Mammalian DNA • 1. Inoculate a deep 96-well plate filled with IB (+ antibiotic marker) with a small amount of bacterial culture. Incubate with shaking at 37˚C • 2. Spin down the cultures and follow the manufacturer’s protocol for the QIAprep • 3. Use 1-5 ul of eluted plasmid DNA as PCR template

  10. Protocol for precipitation and 384 Well Arraying of PCR products • 1. After running reactions on 1% agarose gel and documenting results, add sodium acetate, pH 5.5 and 110 ul room temp isopropanol • 2. Transfer reactions to U-bottom plates,.. tape plates together. • 3. Spin plates at 4.500 rpm for 2 hours • 4. Carefully aspirate solution • 5. Add 100ul 70% EtOH. Spin plates for another hour at 4,500 • 6. Aspirate again and let air dry or dry in a 96 well speed-vac • 7. Allow PCR products to resuspend in 20ul of H2O for at least 18 hours • 8. Transfer products to 384 -well printing plates • 9. Dry plates down in speed-vac and resuspend products in 3X SSC • 10. Let plates resuspend overnight before printing.

  11. Printing Approaches • Non - Contact • Piezoelectric dispenser • Syringe-solenoid ink-jet dispenser • Contact (using rigid pin tools, similar to filter array) • Tweezer • Split pin • Micro spotting pin

  12. Micro Spotting pin

  13. Practical Problems • Surface chemistry: uneven surface may lead to high background. • Dipping the pin into large volume -> pre-printing to drain off excess sample. • Spot variation can be due to mechanical difference between pins. Pins could be clogged during the printing process. • Spot size and density depends on surface and solution properties. • Pins need good washing between samples to prevent sample carryover.

  14. Post Processing Arrays • Protocol for Post Processing Microarrays • Hydration/Heat Fixing • 1. 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 side • 3. On back of slide, etch two lines above and below center of array to designate array area after processing • 4. Pour 100 ml 1X SSC into hydration tray and warm on slide warmer at medium setting • 5. Set slide array side down and observe spots until proper hydration is achieved. • 6. Upon reaching proper hydration, immediately snap dry slide • 7. Place slides in rack.

  15. Surface blocking • 1. Store succinic anhydride in vacuum dessicator until ready for use. • 2. Measure 335 ml 1-methly-2-pyrrolidinone into designated clean dry slide dish with stir bar • 3. Dissolve 5.5 g succinic anhydride completely • 4. IMMEDIATELY after succinic anhydride dissolves, mix in 15 ml 1M NaBorate pH 8.0 and submerge slides in solution. Shake evenly under level of solution. • 5.Soak slides in solution on shaker for 15’ • 6. Before 15’ incubation is done, reduce heat on boiling water so temp is approx 95C and no more bubbles are present. Drain excess blocking solution off slides and transfer slide rack to boiling water and incubate for 1’30” • 7. Transfer rack to dish of 95% EtOH and plunge 5X. Spin down on tabletop. • 8. Arrays may be used immediately or stored for future use.

  16. Isolating Nucleic Acid: “RNA, Membranes, and Tumors” • Protocol for Total RNA isolation in S. Cerevisae • Modified FastTrack Protocol for Yeast Poly-A RNA Isolation • Protocol for Poly-A Isolations • Revised Protocol for FastTrack mRNA extraction from Human Cells • Tumor mRNA isolation • Gradient-based membrane-bound Polysome Protocol • Protocol for Immunoprecipitation of Chromatin from Fixed Yeast Beadbeater Method

  17. Protocol for Total RNA Isolation in S. Cerevisae • 1. Spin down cells (about 250ml at OD600=0.5). Dump supernatant. • 2. Resuspend in 12 ml of AE Buffer. Transfer to Oak Ridge phenol resistant 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 rotor • 7. Dump supernatant into pre-spun 50 ml PhaseLock tube.Add 15 ml chloroform and shake to mix (…ctd)

  18. 8. Spin down 10’ at 3,000 rpm in table-top centrifuge • 9. Dump supernatant into new oak Ridge tube • 10. Add 1/10 volume 3M NaAcetate and equal volume of room temperature isopropanol • 11. Spin down 35’-40’ at 12,000 rpm in SS34 • 12. Wash with 70% EtOH, resuspending pellet, spin again 20’ at 12,000 rpm • 13. Dump off EtOH. Dry pellet in vacuum oven briefly • 14. Resuspend in 500ul water • 15. Quantitate via spec and run 1ug on 1% agarose gel • 16. Store total RNA in -80˚C • Protocol for Poly-A Isolations more complex: 18 steps.

  19. Labelling Nucleic Acid • Protocol for Reverse transcription and Amino-allyl Coupling of RNA • Preparation of Fluorescent cDNA Probe from Human mRNA (alternate protocol) • Modified Eberwine (“ANTISENSE”) RNA Amplification Protocol • Protocol for labeling Genomic DNA for Microarrays: Version 1 • Genomic DNA Labeling Protocol • Round A/B DNA Ampification Protocol

  20. Preparation of Fluorescent cDNA Probe from 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 total volume of 15 ul (x 2) • 2. Heat to 70 ˚C for 10 min and cool on ice • 3. Add 15 ul of reaction mixture each to Cy3 and Cy5 reactions (5X first strand buffer, 0.1M DTT, unlabeled dNTPs, Cy3 or Cy5, Superscript II • 4. 5X first strand buffer: 250 mM Tris-HCl, 375 KCl, 15mM MgCl2 • 5. Incubate at 42 ˚C for 1.5-2 hrs • 6. Degrade RNA by addition of 15ul of 0.1N NaOH, and incubate at 70 ˚C ……(ctd)

  21. 7. Neutralize by addition of 15 ul of 0.1N HCl, and bring the volume to 500 ul with TE • 8. Add 20 ug of Cot1 human DNA • 9. Purify probe by centrifuging in a Centricon micro-concentrator • -------------------------------------------------------------------------------------- • 10. Combine the separate concentrated probes (Cy3 and Cy5) into a fresh Centricon, bring to a volume of 500 ul with TE and concentrate again • 11. Add 1 ul of 10ug/ul polyA RNA and 1 ul of 10ug/ul tRNA • 12. Adjust volume to 9.5 ul with distilled water • 13. For final probe preparation add 2.1 ul 20XSSC and 0.35 ul 10% SDS. Final probe volume can be adjusted to between 12 ul and 15 ul. • 14. Denature probe by heating for 2 min at 100 ˚C, and incubate at 37 ˚C for 20-30 min • 15. Place on array under a glass cover slip • 16. Hybridize at 65 ˚C for 14 to 18 hours in a custom slide chamber with humidity maintained by a small reservoir of 3XSSC • 17. Wash arrays by submersion and agitation for 2-5 min in 2X SSC with 0.1%SDS followed by 1X SSC and 0.1X SSC • 18. Spin dry by centrifugation for 2c min on a slide rack in a tabletop centrifuge at 650 rpm for 2min

  22. Hybridization • Humidity • Temperature • Formamide • (Lowers the Tm) 3XSSC HYB CHAMBER ARRAY LIFTERSLIP SLIDE LABEL SLIDE LABEL

  23. Hybridization Chamber

  24. Protocol for Array Hybridization • 1. Prepare probe as described at the end of the labeling protocol • 2, Set slide in hybridisation chamber • 3. Clean a lifterslip with EtOH and Kimwipes. Place slip on array using either fingers or forceps • 4. Boil probe for 2 min at 100 ˚C. Let cool 5-10 min at room temp. • 5. Slowly inject the probe under one corner of the cover slip until the array surface is covered. Continue to apply remaining probe at the other corners. • 6. Tightly screw down chamber lid and carefully place chamber in a 63˚C water bath, • 7. Allow hybridisation to run at least 5 hours but not more than 16 hours.

  25. Array Washing Protocol • Details of 7-stage washing protocol skipped but it is a very important step. • …………... • 8. Try to scan array within hours of washing as the Cy dyes are unstable and will degrade differentially.

  26. Micrograph of a portion of hybridization probe from a yeast mciroarray (after hybridization).

  27. Axon scanner

  28. Summary of analysis possibilities • Determine genes which are differentially expressed (this task can take many forms depending on replication, etc) • Connect differentially expressed genes to sequence databases and perhaps carry out further analyses, e.g. searching for common upstream motifs • Overlay differentially expressed genes on pathway diagrams • Relate expression levels to other information on cells, e.g. known tumour types • Define subclasses (clusters) in sets of samples (e.g. tumours) • Identify temporal or spatial trends in gene expression • Seek roles for genes on the basis of patterns of co-expression • ……..much more • Many challenges: transcriptional regulation involves redundancy, feedback, amplification, .. non-linearity

  29. Part of the image of one channel false-coloured on a white (v. high) red (high) through yellow and green (medium) to blue (low) and black scale

  30. More of the image of one channel

  31. And more again….

  32. Does one size fit all?

  33. Segmentation: limitation of the fixed circle method SRG Fixed Circle Inside the boundary is spot (fg), outside is not.

  34. Some local backgrounds Single channel grey scale We use something different again: a smaller, less variable value.

  35. Quantification of expression • For each spot on the slide we calculate • Red intensity = Rfg - Rbg • fg = foreground, bg = background, and • Green intensity = Gfg - Gbg • and combine them in the log (base 2) ratio • Log2(Red intensity / Green intensity)

  36. Gene Expression Data • Gene expression data on p genes for n samples Slides slide 1 slide 2 slide 3 slide 4 slide 5 … 1 0.46 0.30 0.80 1.51 0.90 ... 2 -0.10 0.49 0.24 0.06 0.46 ... 3 0.15 0.74 0.04 0.10 0.20 ... 4 -0.45 -1.03 -0.79 -0.56 -0.32 ... 5 -0.06 1.06 1.35 1.09 -1.09 ... Genes Gene expression level of gene 5 in slide 4j = Log2(Red intensity / Green intensity) These values are conventionally displayed on a red(>0)yellow (0)green (<0) scale.

  37. The red-green ratios can be spatially biased Top 2.5%of ratios red, bottom 2.5% of ratios green

  38. Another example • .

  39. Back to Affymetrix: obtaining the data • RNA samples are prepared, labeled, hybridized with arrays, and stained as just described • Arrays are scanned and the resulting image analyzed (we omit the details) • Approximately 49 pixels per probe cell are summarized by their 75th %tile, after removal of outer perimeter pixels. This is the probe cell’s intensity • Of interest is to find a way to combine probe cell intensities for a given gene to produce an index of expression – an indicator of abundance of the corresponding target mRNA. We discuss this later.

  40. Affymetrix QA protocols: Overview • Starting RNA QA: look at gel patterns and RNA quantification. • Post hybridization QA : image examination, chip intensity parameters, expressions for control probe sets of various sorts, house keeping genes, percent Present calls.

  41. Starting RNA quality • Gels detect 18S and 28S ribosomal RNA and quality of fragmentation • Spectrophotometric ratio of 260/280 absorbance values used as a quality indicator • Other gel electrophoresis patterns from material at different stages of preparation are used to make qualitative assessments of the RNA samples.

  42. Spectrophotometric analysis of total RNA, Image from Vanderbilt shared resource site http://array.mc.vanderbilt.edu/Pages/VMSR_Info/Sample_submission.htm

  43. Sample quality assessment by agarose gel electrophoresis • For total RNA, look for 18S and 28S bands, see previous slide. • For cDNA, a good sample will produce a smear extending from top to bottom of the gel. • Unfragmented cRNA will also produce a smear running down the gel. • Fragmented cRNA gel should appear as a blob at the bottom of the gel indicating that the cRNA has been successfully fragmented to pieces about 50 bp in length

  44. Affymetrix standards for post hyb and scanning quality assessment:Visual inspection of image • Biotinylated B2 oligonucleotide hybridization: check that checkerboard, edge and array name cells are all o.k. • Quality of features: discrete squares with pixels of slightly varying intensity • General inspection: scratches (ignored), bright SAPE residue (masked out) • Grid alignment

  45. Chip dat file – checkered board – oligo B2

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