1. UVM Microarray Facility Overview and New Capabilities

2. 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

3. 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

4. Current Equipment in the Facility

5. Affymetrix GeneChip System Agilent 2100 Bioanalyzer Nanodrop Spectrophotometer Qubit Spectrofluorometer FastPrep homogenizer BioSpec homogenizer Summary of Equipment in the Facility

6. 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-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

7. 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

8. 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

9. 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

10. Affymetrix Technology

11. 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

12. 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’

13. 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

14. Exon Arrays- (Exon 1.0 ST) 5x10 6 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 probesexon Hun-1 has 6 exons

15. 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].

16. Target Preparation Methods

17. 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 »cRNA b -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

18. Comparsion of NuGEN vs Eberwine 50ng 1 clean-up step 8hr Affy-EberwineNuGEN Ovation 1 ug 2 clean-up steps 3 days

19. 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

20. 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

21. Routine Services Provided

22. 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!!!

23. 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

24. 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

25. Sample Submission

26. 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 data Write paper and publish in Science Get R01 grant and Tenure

27. Microarray Order Forms

28. When your data is complete… Set-up a meeting with Jeff Bond in the Bioinformatics Core for 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 Network through Grant Number P20 RR16462 from the INBRE Program of the National Center for Research Resources (NCRR)”

29. 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

30. - 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 DNA extractions for DNA Mapping chips

31. 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

32. 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 RNA Things are not always as they appear- These look great on the nanodrop…but absorbance at 260 does not tell the condition of the RNA

33. 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, GeneArrays NuGEN-Pico, Pico WT, FFPE, OvationV2- 3’ IVT, Exon, GeneArrays Affy/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

34. RNA of Current study Rin 9 Rin 6 Rin 4 Rin !

35. Special notes on FACS and LCM Please see us for detailed protocols

36. 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

37. 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 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