UVM Microarray Facility

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UVM Microarray Facility Overview and New
Capabilities
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UVM Microarray- background

• Established in 2002 by the Vermont Genetic
  Network
• First project complete 4/7/2003-10 mouse chips
• PersonnelScott Tighe 0.75 FTE and Tim Hunter
  0.4 FTE
• Located in HSRF 305
• Uses the Affymetrix GeneChip System
• Provides wide range of services including
• Genechip Processing
• Training on RNA-DNA techniques
• Recommendation of protocols and reagents
• Guidance on experiment design
• Assistance with grants and publications
• Development of new and unusual protocols

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Utilization
The facility has been well utilized since the
beginning 1360 Genechips for 40 PI at 5
institutes Including LCM, FACS, Tissue, Cells,
Blood, Cartilage, Ligament- to name a few Mouse,
Rat, Human, Bovine, Yeast, Bacteria, Poplar,
Arabidopsis, Drosophila, Pig, Xenopus
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Current Equipment in the Facility
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Summary of Equipment in the Facility

• Affymetrix GeneChip System
• Agilent 2100 Bioanalyzer
• Nanodrop Spectrophotometer
• Qubit Spectrofluorometer
• FastPrep homogenizer
• BioSpec homogenizer

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• Affymetrix GeneChip system
• Primary instrument used for Microarray
• (2) FS-450 Fluidics Stations-allows
• up to 36 samples a day
• 7G high resolution Scanner

• Prokaryotic Expression
• E. coli Genome Array
• P. aeruginosa Genome Array
• S. aureus Genome Array
• B. subtilus Array
• Transcriptome-Expression
• Human Exon Array -400
• Human Gene Array -175
• Mouse Exon Array -400
• Mouse Gene Array -175
• Rat Exon Array -375
• Rat Gene Array-175
• DNA Mapping
• Human SNP DNA Mapping Array 5.0/6.0- 175/300

Eukaryotic-Expression Arabidopsis Genome Array
Bovine Genome Array -250 Barley Array C.
elegans Genome Array Canine Genome 2.0 Array
Chicken Genome Array Citrus Genome Array
Cotton Genome Array Drosophila Genome
2.0-250 Human Genome Arrays 400/250 Maize
Genome Array Medicago Genome Array Mouse Genome
Arrays -250/400 Plasmodium/Anopheles Array
Poplar Genome Array Porcine Genome Array Rat
Genome Arrays -375 Rhesus Genome Array Rice
Genome Array Soybean Genome Array Sugar Cane
Genome Array Tomato Genome Array Vitis vinifera
Genome Array Wheat Genome Array Xenopus
tropicalis Array Xenopus laevis Arrays Yeast
Genome Arrays Zebrafish Genome Array

• Microarray GeneChips
• Eukaryotic
• DNA SNP Mapping,
• 3 Expression, Exon, Gene,
• tiling, promoter
• Prokaryotic-Expression only
• Single Use disposable

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• Agilent 2100 Bioanalyzer
• Used to test the integrity of
• total RNA
• mRNA
• cRNA
• cDNA
• miRNA
• RIN scores (1-10)
• Used for semi-quantitation of micro-dissected RNA
  or Low recovery RNAs

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Nanodrop Spectrophotometer Used for
quantitating RNA and DNA Range of 3ng to
3500ng/ul
Qubit Spectrofluorometer Used for quantitating
from 50pg- 100ng RNA and DNA Uses fluorescent
intercalating dyes Good when interfering
compound skew nanodrop Good for LCM or FACS
Require a standard curve
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• FastPrep System and Mini-bead beater
• Automated, fast, RNase-free homogenizers
• Uses screw cap 2ml, 15ml, and 50ml tubes
• Optional abrasive for homogenizing tough tissue
• Excellent for bacterial extractions and difficult
  tissues

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Affymetrix Technology
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Affymetrix Microarray

• Disposable plastic cartridge-based microarray
• 1 cm x 1cm glass window with DNA oligos bond to
  the surface
• Each DNA oligo is 25bp and represents a part of a
  gene sequence

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GeneChip Types- Gene Expression
3 Arrays

• Target generated by Eberwine synthesis approach
• 11 Probes are designed to the first 600-800bp of
  a gene
• Synthesis reactions can produce truncated cRNA
  product
• No data on alternative splicing
• Still popular

Hun-1 has 6 exons
U133A U133 2.0 Plus 430A 2.0 430
2.0 3
Perfect match oligos
1bp-Mis-match control oligos
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Phillips J, Eberwine JH. (1996) Antisense RNA
Amplification A Linear Amplification Method for
Analyzing the mRNA Population from Single Living
Cells. Methods. 10(3)283-288.
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New GeneChip Types- Gene Expression

• GeneArrays- (GeneArray 1.0 ST)
• -764,000 probes for 28,869 genes
• - 26 probes per gene ave with 1 probe per exon
  min
• -some data on alternative splicing
• -Less expensive
• -More expensive Target prep-Random amplification
• -Overall cheaper than 3 Array

---Human, Rat, Mouse
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New GeneChip Types- Gene Expression

• Exon Arrays-(Exon 1.0 ST)
• 5x106 probes against 1 million exon clusters
  representing 150,000 transcript variants
• Minimum 4 probes per exon
• Definitive data on alternative splice variants
• More expensive GeneChip
• More expensive Target prep-Random amplification

---Human, Rat, Mouse
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GeneChip Types-DNA Mapping

• Applications include CGH,SNP, LOH, NP, and CNV
• Requires 250ng of gDNA
• Formats include 100k 500k, 1 million
• Requires a reference sample or replicates
• Contains over 7.5 million probes per chip

CGH of Chromosome 5 of Skin tumor compared to a
blood reference. Data indicates a 50 mb deletion
to 1 copy in q-arm between q21.3 and q33.1 106Mb
to 156 Mb.
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Target Preparation Methods
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Target Synthesis Methods

• 3 expression GeneChip Arrays
• Eberwine-based or the Affy Method (requires 1-3
  ug)
• T7 Oligo-dT primer
• Generate ds-cDNA
• Biotinylated cRNA synthesized via IVT reaction
• cRNAb-DNA hybrid on GeneChip
• Many companies sell the same system
• NuGENs Ribo-SPIA Technology (requires 20-80ng)
• Both Oligo dT and Randomers
• Generates amplified cDNA using RNase H, a
  chimeric RNA/DNA primer, and polymerase.
• cDNA-DNA hybrid on GeneChip end labelled with
  Tdt
• Choice for FACS and LCM derived RNA
• As little as 1ng/ul of RNA required for the pico
  version

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Comparsion of NuGEN vs Eberwine
Affy-Eberwine
NuGEN Ovation
50ng 1 clean-up step 8hr
1 ug 2 clean-up steps 3 days
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Target Synthesis Methodscont

• Exon and GeneArray ST Arrays
• Affy method (100ng-2ug)
• Requires ribosomal reduction step-Noisey
• Variable data-many steps-many clean ups
• 5 day prep
• NuGEN Pico WT Method (1 ng -40 ng)
• No Ribosomal reduction required
• Good SN
• Very little variation and better call rates
• 2 day prep

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ExpressArt by AMP-TEC a New Option?
Target Synthesis Methodscont

• useful for degraded RNA presently being
  evaluated
• Uses a random Trinucleotide primer
• Combined with Affymetix and Enzo IVT reagents
• Exon, GeneArrays, and standard 3 Arrays
• Initial studies look good
• 3 Arrays (requires 500ng)
• Uses a trinucleotide primer to generate cDNA
• Standard Enzo IVT to generate biotinylated cRNA
• EXON and GENEARRAYs (requires 500ng)
• No ribosomal reduction
• High call rates
• Excellent fidelity

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Routine Services Provided
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Routine Services Provided

• Affymetrix GeneChip processing
• 3 IVT expression arrays
• 500 K and 5.0 DNA mapping arrays
• Exon arrays-Since Sept 2008
• GeneArrays-Since Sept 2008
• Target Preparation Method
• Moving to NuGEN SPIA for all chips 3, Exon,
  Gene
• Affy-Eberwine for past-continuing projects-By
  request
• Please ask and be specific on order forms
• ExpressArt for degraded RNA by special
  arrangement only
• Pending results from our study!!!

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Routine Services Providedcont

• RNA assessment with Bioanalyzer
• Total RNA (500pg-500ng/ul)
• mRNA (500pg-500ng/ul)
• miRNA (1-20 ng/ul)
• cDNA, cRNA, PCR product
• Training on RNA handling
• RNA and DNA Extractions-By the DNA Facility
  Meghan
• Microarray experimental design multidisciplinary
  approach
• Protocol development
• Sample reagents
• Troubleshooting

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Summary of New Services and Changes

• Price reduction on GeneChips
• We now get teir 4 pricing just as Harvard or MD
  Anderson
• 50/genechip cost reduction (ave)
• Exon arrays
• Gene Arrays
• Micro and Small RNA assessment
• Qubit spectrofluorometer
• New target preparation method
• Nugen preparations require only 40-60 ng/sample
• Amp-tec for moderately degraded RNA-pending
  results of our study

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Sample Submission
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Sample Submission

• Initial consult with Bioinformatics, microarray,
  and PI staff
• Isolate RNA at gt20ng/ul or DNA at gt50ng/ul
• Submit RNA assessment form and 2ul of RNA
• Wait for results (24hr)
• Submit 100ng of good RNA or 250 ng gDNA
• RNA may have an RNase inhibitor if you wish
• Submit target preparation form

No stocks please- I throw leftovers out at 6
months
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Microarray Order Forms
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When your data is completeSet-up a meeting
with Jeff Bond in the Bioinformatics Corefor
your data analysis. Remember that data analysis
can be real work and be prepared to spend some
time at it.January Seminar BioinformaticsReme
mber to site the Microarray facility and VGN in
your acknowledgements because the labor is
subsidized by VGN. See the VGN website for a
cut and paste version.This publication was
made possible by the Vermont Genetics
Networkthrough Grant Number P20 RR16462 from the
INBRE Program of theNational Center for Research
Resources (NCRR)
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RNA Protocols

• Trizol-RNeasy Hybrid (RNeasy Lipid)
• Extract in trizol
• add BCP and spin
• transfer aq layer 2vol 100 etoh to a RNeasy
  column
• Follow SOP of Qiagen but wash 1x RW1, 2x times
  RPE
• Good for high interference materials
• May have interfering trizol pk on nanodrop
• Trizol LS great for FACS
• RNeasy Mini or Micro
• Trizol Only-When have significant RNA yield
• MicroRNAs
• Be sure your method recovers small RNAs
• Trizol is ok
• Qiagen and Ambion has kit designed with Trizol
  and silica column
• Other kits are available???

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DNA extractions for DNA Mapping chips
-DNazol When DNA is expected to be
abundant Recovers small DNAs Precipitation
required -QiaAmp Micro Kit Good for small yield
material FACS-LCM -Bring to DNA lab-Meghan will
do them for you -Run gel and check on nanodrop
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Quality Control of RNA

• Measuring your RNA on the Nanodrop
• Look carefully at the trace!
• Can not distinguish DNA from RNA
• Can not distinguish degraded RNA from good RNA
• Quantitative interferences can lead to
  questionable downstream results

Good RNA
RNA prep with mostly gDNA
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Degraded RNA
Quality Control of RNA

• Things are not always as they appear- These look
  great on the nanodropbut absorbance at 260 does
  not tell the condition of the RNA

Good RNA
Valuable data from the Nanodrop includes 260230
(ie salt and small interfering molecules)
..Recommended value ..gt1.0 260280 (amount of
contaminating protein).Recommended value
....gt1.7 Protein absorbs at
280/-5nm 260320 (large molecules-agarose)
.... gt10 ????
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Recent Studies in the Facility

• Freeze thaw cycling of RNA
• RNA that is clean and RNase-free is very
  stable-freeze thawed 4x
• 32 hours at room temp and 64 hours at -20 for
  four cycles
• No changes

• Converting cDNA back to polyA total RNA for
  microarray
• Use Genisphere reagents
• Started with superscript RT cDNA from Oligo dt
• Uses polyT tailing of cDNA
• Synthesis of ds T7 protomer using polyA-T7
  primer and Klenow
• IVT produces a new sRNA strand
• Attachment of poly A using PAP enzyme
• In progress nowgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt
• Evaluation of commercial reagents and protocols
  to amplify mRNA in HBR RNA samples at various
  stages of degradation as measured by microarray
  and qPCR
• AMPTec-ExpressArt- 3 IVT, Exon, GeneArrays
• NuGEN-Pico, Pico WT, FFPE, OvationV2- 3 IVT,
  Exon, GeneArrays
• Affy/Invitrogen- 3 IVT, Exon, GeneArrays

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RNA of Current study
Rin 9 Rin 6 Rin 4 Rin !
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Special notes on FACS and LCMPlease see us for
detailed protocols
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FACS sorted cells and RNA

• 1 FACS must be RNase-free by bleach and other
  treatments
• 2 Bacterial contamination in sheath tanks and
  dip tubes can cause RNases
• 3 Hold back cells check viability- extract RNA
  as a pre-sort control
• 4 Add Superase to sort tube and pre-sort tube
  containing cells
• 5 Use RNase-free tubes to sort
• 6 Sort into an exact volume of ice cold
  extraction reagent
• Trizol LS or STD not recommended for limited
  cells numbers
• RLT buffer RNeasy Micro
• Media or buffer with or without Superase
• 7 After sorting measure the total volume and add
  the necessary volume of reagent to obtain the
  correct ratio of extraction reagent to sort
  liquid
• 8 Consider acceptable sort volumes

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LCM and RNA

• Test tissue section before starting by
  aseptically removing and extracting a small
  section. DO NOT LET FROZEN TISSUE THAW!
• FFPE tissues often yield no usable RNA and
  testing its condition before the start is a good
  idea
• Know what level of degradation your downstream
  application can tolerate
• Fresh frozen tissues perform well
• Use RNase Zap-ETOH treated microtome with new
  blade during sectioning
• Use RNase free slides, tweezers, materials
• Scape a small section from the prepared slide
  and extract as a controlPrepare all staining
  and dehydration reagents from RNase-free reagents
  and DEPC waterCollect one LCM cap from large
  area of cells as a control-non-specific
  cellsCollect target cells and transfer cap to
  tube containing extraction agent-vortexExtract
  immediatelyWe have had good luck with RNeasy
  micro and Pico Pure kitsOmit DNase step to
  increase recovery when allowableExtract several
  caps into one extract buffer or several extract
  buffers and combine to increase yield