UVM Microarray Facility Overview and New Capabilities

3. Utilization

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

8. Nanodrop Spectrophotometer Used for quantitating RNA and DNA Range of 3ng to 3500ng/ul

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

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

14. 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

15. GeneChip Types-DNA Mapping 

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 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

18. Comparsion of NuGEN vs Eberwine

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

22. 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!!!

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

26. Sample Submission 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 Corefor your data analysis. Remember that data analysis can be real work and be prepared to spend some time at it.January Seminar: BioinformaticsRemember 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)”

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???

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

32. Degraded RNA

33. Recent Studies in the Facility

34. RNA of Current study

35. Special notes on FACS and LCMPlease 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 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