UVM Microarray Facility Overview and New Capabilities.
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Transcript of UVM Microarray Facility Overview and New Capabilities.
UVM Microarray- background
• Established in 2002 by the Vermont Genetic Network• First project complete 4/7/2003-10 mouse chips
• Personnel–Scott 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
Utilization
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2003 2004 2005 2006 2007 2008
PicoChips
NanoChips
GeneChips
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
• Affymetrix GeneChip System• • Agilent 2100 Bioanalyzer
• Nanodrop Spectrophotometer
• Qubit Spectrofluorometer
• FastPrep homogenizer• BioSpec homogenizer
Summary of Equipment in the Facility
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
• Human 10/100/500K SNP DNA Mapping -$150
• Human Mitochondrial Array • SARS Array • Human Promoter-Tiling
Microarray GeneChips•Eukaryotic
•DNA SNP Mapping,• 3’ Expression, Exon, Gene, tiling, promoter
Prokaryotic-Expression only•Single Use disposable
Eukaryotic-ExpressionArabidopsis Genome Array Bovine Genome Array -$250Barley ArrayC. elegans Genome Array Canine Genome 2.0 Array Chicken Genome Array Citrus Genome Array Cotton Genome Array Drosophila Genome 2.0-$250Human Genome Arrays $400/250Maize Genome Array Medicago Genome Array Mouse Genome Arrays -$250/400Plasmodium/Anopheles Array Poplar Genome Array Porcine Genome Array Rat Genome Arrays -$375Rhesus 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
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 RNA’s
Nanodrop Spectrophotometer
Used for quantitating RNA and DNARange of 3ng to 3500ng/ul
Qubit SpectrofluorometerUsed for quantitating from 50pg- 100ng RNA and DNAUses fluorescent intercalating dyesGood when interfering compound skew nanodropGood for LCM or FACS Require a standard curve
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
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
GeneChip Types- Gene Expression
U133A
U133 2.0 Plus
430A 2.0
430 2.0 3’
Perfect match oligos
1bp-Mis-match control oligos
Hun-1 has 6 exons
• 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”
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.
3’ Arrays
5’
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
probe exon
Hun-1 has 6 exons
New GeneChip Types- Gene Expression
---Human, Rat, Mouse
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
New GeneChip Types- Gene Expression
---Human, Rat, Mouse
4 probes exon
Hun-1 has 6 exons
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].
Target Synthesis Methods
• 3’ expression GeneChip Arrays– Eberwine-based or the Affy Method (requires 1-3 ug)
» T7 Oligo-d[T] primer» Generate ds-cDNA» Biotinylated cRNA synthesized via IVT reaction» cRNAb-DNA hybrid on GeneChip» Many companies sell the same system
– NuGEN’s Ribo-SPIA Technology (requires 20-80ng)» Both Oligo d{T} 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
Comparsion of NuGEN vs Eberwine
50ng 1 clean-up step 8hr
Affy-Eberwine NuGEN Ovation
1 ug 2 clean-up steps3 days
• 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 S:N» Very little variation and better call rates» 2 day prep
Target Synthesis Methods…cont
ExpressArt ® by AMP-TEC: a New Option?
– 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
Target Synthesis Methods…cont
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!!!
Routine Services Provided…cont
• 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
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
Sample Submission
No stocks please- I throw leftovers out at 6 months
Initial consult with Bioinformatics, microarray, and PI staff
Isolate RNA at >20ng/ul or DNA at >50ng/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
Wait for awesomely great super dataWrite paper and publish in Science
Get R01 grant and Tenure
When your data is complete…
Set-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: Bioinformatics
Remember 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 the
National Center for Research Resources (NCRR)”
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
– MicroRNA’s• Be sure your method recovers small RNA’s• Trizol is ok• Qiagen and Ambion has kit designed with Trizol and silica
column• Other kits are available???
260 272 RNA Trizol
-DNazol
When DNA is expected to be abundant
Recovers small DNAsPrecipitation required
-QiaAmp Micro KitGood for small yield material
FACS-LCM
-Bring to DNA lab-Meghan will do them for you
-Run gel and check on nanodrop
DNA extractions for DNA Mapping chips
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
Degraded RNA
Valuable data from the Nanodrop includes
260:230 (ie salt and small interfering molecules) ……..Recommended value …..>1.0
260:280 (amount of contaminating protein)………….Recommended value ………....…>1.7
Protein absorbs at 280+/-5nm
260:320 (large molecules-agarose)……………………………………………………………..…………….. >10 ????
Quality Control of RNA
Good RNAThings are not always as they appear- These look great on the nanodrop…but absorbance at 260 does not tell the condition of the RNA
Recent Studies in the Facility
Converting cDNA back to polyA total RNA for microarray•Use Genisphere reagents •Started with superscript RT cDNA from Oligo d[t]•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 now>>>>>>>>>>>>>>>>>>
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, GeneArraysNuGEN-Pico, Pico WT, FFPE, OvationV2- 3’ IVT, Exon, GeneArraysAffy/Invitrogen- 3’ IVT, Exon, GeneArrays
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
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
9] Extract immediately-do not store cells in extraction buffer at -80
10] DNase-treatment causes RNA losses using RNeasy
LCM and RNATest 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 sectioningUse RNase –free slides, tweezers, materials Scape a small section from the prepared slide and extract as a control
Prepare all staining and dehydration reagents from RNase-free reagents and DEPC water
Collect one LCM cap from large area of cells as a control-non-specific cells
Collect target cells and transfer cap to tube containing extraction agent-vortex
Extract immediately
We have had good luck with RNeasy micro and Pico Pure kits
Omit DNase step to increase recovery when allowable
Extract several caps into one extract buffer or several extract buffers and combine to increase yield