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Project Documentation & Protocols: Maize Gene Discovery Project: Microarrays: Protocols Contents: Index | Progress | Controls | Libraries | Arrays | Data | Protocols | Ordering | Links | FAQs Your Microarrays Your Spiking Controls Steps for Hybridization, Data Aquisition, and Data Analysis Your Microarray Format Navigating Your Microarray Preliminary Analysis of your Microarray Data 2. Preparation of Spiking Controls 3. Preparation of Total RNA (using TRIzol Reagent-GIBCOBRL) 4. Preparation of polyA+ mRNA (using Dynabeads Oligo (dT)25--Dynal) 5. Direct Labeling of polyA+ mRNA 6. Hybridization of Labeled Targets to Microarrays Instructions to Microarray UsersBEFORE HANDLING SLIDES Take care to handle slides only by the labels or edges to avoid smearing or leaving residues from your fingers on the slides. Your slides have been shipped to you in a polyfoam slide case. They should be stored dry (in the case, sealed within a plastic bag containing desiccant) at room temperature no longer than 12 months. Inside the case, slides may be shipped with 1 or 2 slides per slot depending on the number of slides you ordered. If there are 2 slides per slot, then the slides have been shipped back-to-back and must be separated and carefully reoriented before use so that the label side of both slides is up. The DNA is printed on the side with the label-If you can read the label, you have the correct side up and it is safe to set your slides down on a clean, dry surface. YOUR MICROARRAYS Check the label of your slides for EST library and printing format information. The slide number provides the EST Library number /Microarray Format /Print Number / Slide number (see example next page). Click on the information button on the Microarray Elements Search page for information on the preparation and format of your slides. YOUR SPIKING CONTROLS We have enclosed several spiking controls on paper filter disks. STEPS FOR HYBRIDIZATION, DATA ACQUISITION, AND DATA ANALYSIS Please read the overview provided below before beginning your work. Note that you must have access to certain equipment before proceeding. Protocols for all steps are available on this webpage and on the University of Arizona webpage -see Maize Gene Discovery Protocols. We recommend that you start by following our DNA immobilization protocol that has been optimized for the slide coating on your slides. If you are a beginner, we encourage you to start with the protocols working in our lab. If you are not, we certainly encourage you to follow your own protocols for steps other than DNA immobilization and contribute back to us what works best for you. 1. Immobilization of DNA on slides. Your slides have been sent to you WITHOUT immobilization of the DNA. You will need to MARK THE BOUNDARIES OF YOUR ARRAY before immobilization and proceed with the protocol before you begin handling your slides to any extent-slides may remain in the slide box without DNA immobilization. You will need access to a UV cross-linking instrument. We suggest you try rehydration for 10 seconds on your first slide(s) and add time (10-20 seconds) if you feel you need larger spots-be careful or spots will run together.
2. Preparation of mRNA. For each hybridization, you will need ~4 µg of polyA+ mRNA for each treatment-if you plan to hybridize a single slide with mRNA from two different sources (e.g. a control and a treatment), you will need a total ~8 µg of mRNA per slide. We routinely isolate total RNA using TRIzol (GibcoBRL) and purify using DynaBeads Oligo (dT)25 (Dynal) according to manufacturers instructions.
3. Preparation of Spiking Control. If you plan to use one or more spiking controls, you will want to prepare them prior to direct labeling. A small aliquot of the spiking control can be added to the labeling reaction mix for each treatment. 4. Direct Labeling of polyA+ mRNA. We routinely incorporate label directly as Cy3- or Cy5-labeled dUTP (Cy3-, Cy5-dUTP from Amersham Pharmacia catalog numbers PA53022 and PA55022), using Sigma.s AMV-RT Kit (catalog number STR1-KT) according to manufacturers instructions. You will need access to the reagents above as well as to waterbaths at various temperatures.
5. Hybridization. We follow a modified version of the protocol recommended by the manufacturer for Sigma Screen Silane slides (Modified from Sigma Technical Bulletin MB-745 November 1999-Product No: A7718 ArrayHyb Buffer). You will need to purchase certain reagents (see Protocol) as well as have access to a reliable 60-65 C incubator. 6. Data Acquisition. You will need reliable access to a Microarray slide reader. If you are using a fluorescent dye to label your hybridization mix, your slides should be scanned within 8 hours after hybridization.
7. Data Analysis. Check the slide label for the Microarray format used to print your slide. Microarray Element Tables are provided on our webpage for each microarray format used for printing. A search table allows you to quickly identify each printed spot by location and provides EST and BlastX identification. A complete Microarray Element Table for each Microarray Format can downloaded in text format for import into spreadsheets and data analysis programs. You will need access to data analysis software-We use a program called ImaGene (BioDiscovery) that must be purchased. However data analysis software is often available as part of the slide reader software package and some programs are available as freeware on the internet. Your Microarray Format The label of your slides provides information on the EST library and printing format used to make your slides (note there may be more than one printing format for an EST Library). The slide number provides the EST Library number/Microarray Format /Print Number/Slide number (see example below). You can click on the information button below the Microarray Element Table on our webpage for specific information on the preparation and format of your slides. Navigating your Microarray Each slide is composed of eight subarrays. When a slide is oriented with the label at the bottom (see below), the subarrays are positioned in 4 Rows and 2 columns. The location of each spot in a subarray is given by 4 variables: Subarray Row, Subarray Column, Row within Subarray, Column within Subarray. Once you know the location of a printed spot, you can use the information in the Microarray Element Table to provide EST and BlastX identification. A complete Microarray Element Table for each microarray format can downloaded in text format for import into spreadsheets and data analysis programs. 
Preliminary Analysis of your Microarray Data Recommended protocols for immobilization of DNA on slides, preparation of spiking controls and RNA samples, labeling targets, and hybridization are given in the next section. After completion of your hybridization, you will need to scan your slide immediately and acquire an image of your hybridized microarray. Data analysis begins with the use of software to find spots and acquire signal intensity and background information for each spot. You will need access to data analysis software. Typically data analysis software comes with or is recommended for use with your scanner. Free data analysis software can also be found on various Microarray websites-we do not provide data analysis software. A. Obtaining signal intensity data, obtaining a key to spot identity, and merging data and identity files
Example: The 614 microarray has 8 subarrays printed in four rows and 2 columns (see Navigating Your Microarray for an illustration defining rows and columns). Within each subarray, the spots are printed in 45 rows and 45 columns. When spot finding, keep in mind there are several blank spaces in the last row of each subarray-this can be verified by consulting the Microarray Element Search Table. Select 614 as the Array type, 45 for Row # within Subarray, and blank for Stanford ID.
B. Computing Signal Intensity and Background, Data filtering, Calculation of Preliminary Statistics, Estimating Reproducibility, and Representing Data Please keep in mind that there is currently no consensus on how best to analyze or represent microarray data. The following discussion proposes several considerations and data analysis steps-keep in mind that microarray data analysis is a constantly evolving field and our recommendations are likely to change. We recommend that you consult the literature for the protocols that are the most up-to-date and relevant to your experiments.
Once again, we recommend that you consult the literature for the analysis protocols that are the most up-to-date and relevant to your experiments.
Protocols1. IMMOBILIZATION OF DNA ON SIGMA SILANE SLIDES (these slides need backing at 80 C immediately after printing, which is done at the microarray facility at U of A) 1. Mark the boundaries of the array on each slide using a diamond scriber (array will become invisible after post-processing). Be careful not to press so hard slides break. 2. Hold the slide (face down) over a 42 °C water bath for 5-10 seconds to the re-hydrate spots. Note with high density printing (center to center spacing 200 um or less), do not exceed 10 seconds re-hydration time (pre-warming the slides DNA-side up for 3-5 seconds over a 65 C heat block will reduce the chances for over re-hydration)
3. Snap-dry each slide (DNA side up) on a 65 °C heat block for 3-10 seconds until the humidity is gone. Repeat sets 2 & 3 three times. 4. Place arrays in a slide rack and UV crosslink DNA to glass with 65 mJ of 254 nm UV light. (We use a Sigma Humidity Chamber {Sigma H6644} to hold the slides. We use a Fisher Scientific FB UVXL-1000 UV Cross Linker set to 650 x 100 uJ/cm2 for crosslinking DNA). 5. Incubate slides for ~ 5 minutes in 1% SDS on orbital shaker. This step removes the unbound nucleic acids from the arrays and prevents them from binding elsewhere. (We use Sigma staining chambers and racks {Sigma S6141} for all rinses. Make sure the volume in chamber completely covers slides while shaking). 6. Gently plunge the slide rack in boiling water for 2 min to denature the DNA. 7. Rinse slides by plunging rapidly 10-20 times in a 100% ethanol bath (use a clean staining chamber and transfer slides to ethanol still in same staining rack). 8. Transfer slides to 50 ml disposable centrifuge tubes and spin slides at 50-100 x g for 2-5 min. This step must be done quickly and carefully to avoid streaking and smearing arrays. 9. Use arrays immediately or store in a slide box (room temperature, with desiccant). 2. Preparation of Spiking Controls (Modified from Hong Wang-University of Arizona 8/1/99)we recommend to use National spiking controls (SP1-SP6) which is included in our arrays. Spiking controls may be used, if desired, to compare the efficiencies of the labeling reactions in experiments where microarrays are probed with RNAs from two populations (e.g. control and treatment populations). We will supply different spiking controls on paper filter disks on request (see bottom of this protocol for a description of the controls). These same spiking controls have also been printed on the arrays you received. Instructions to prepare spiking controls appear below. If you choose to use a spiking control, you would introduce it into both of your RNA samples in equal concentration prior to labeling. Care must be taken to minimize pipetting errors so that hybridization efficiency of the spiking controls will accurately reflect efficiencies of the two labeling reactions. Frequently, several spiking controls are introduced into both labeling reactions, such that each control is at a different concentration (e.g. Control 1, 2, 3, 4, 5 at 2.0, 1.0, 0.2, 0.1, and 0.02 ng, respectively). Preparation of your controls will involve standard transformation and large-scale plasmid preparation, preparation of RNA by induction of the T7 promoter, and RNA purification. Use of a spiking control is not necessary for analysis of Microarray data. 1. Reconstitute plasmid DNA (~50 ng) on the filter paper disk by placing the disk in 25 µl TE and vortexing. 2. Aliquot ~10 ng of plasmid DNA for transformation. Store remaining plasmid DNA at -20 °C. 3. Transform E.coli using standard laboratory transformation protocols. Select for transformants LB plates with appropriate antibiotic (see control table). 4. Check clones by preparing a 500 µl miniprep, digesting with the restriction enzyme , and analyzing for single bands on a 1.0 % agarose gel. 5. Prepare a large-scale plasmid preparation (50 ml culture) of the confirmed clones with PEG purification. Measure the plasmid concentration. 6. Aliquot ~10 µg of plasmid DNA and repurify plasmid using a Qiagen PCR purification column. Elute the DNA in 30 µl Tris-HCl (10 mM, pH 8.0). Store remaining plasmid DNA at -20 °C. 7. Digest the total 30 µl with the restriction enzyme in a 100 µl reaction mix. After 2 hours, aliquot 1-2 µl and run a 1.0 % agarose gel to check the digestion. 8. Extract the linearized plasmid once with an equal volume of phenol:chloroform and then 3 times with an equal volume of chloroform. 9. Add 0.1 volume of 3 M sodium acetate (ph 5.2) and 2 volumes of ethanol. Mix thoroughly and precipitate at -20 °C for ~ 2hrs. 10. Spin at 12,000 x g at 4 °C for 15 min. 11. Wash pellet with 70% ethanol and dry with SpeedVac. Resuspend the linearized plasmid in 10 µl of TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Adjust final concentration to ~about 1.0 µg/µl. 12. Prepare transcription reaction mix. In a new Eppendorff tube, add 20 µl of 5X transcription buffer, 4 µl linearized plasmid (1.0 µg/µl), 16 µl rNTP mix (2.5 mM each), 4 µl DTT (0.75 M), 1.5 µl RNase Block (40 units/µl), and 54 µl of DEPC treated water. Mix by tapping the tube gently. 13. Add 0.8 µl of T7 RNA polymerase (50 Units/µl). Mix by tapping the tube gently. 14. Incubate at 37 °C for 1-1.5 hours. 15. Add 2 µl RNase free DNase I and mix well. Incubate at 37 °C for 15 min. 16. Extract the reaction once with and equal volume of phenol:chloroform and 3 times with an equal volume of chloroform. 17. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. Mix thoroughly and precipitate at -20 °C for ~ 2hrs. 18. Spin at 12,000 x g at 4 °C for 15 min. There should be a large white pellet at the bottom of the tube. 19. Wash the pellet with RNase free 70 % ethanol and dry by SpeedVac. Resuspend the pellet in 20 µl 1X TE (RNase free). 20. Take 1 µl to run a formaldehyde gel to check the transcript integrity. Measure the concentration by spectrophotometer. 21. Use transcripts immediately or store at -70 °C. Note, transcript quality diminishes significantly with each freeze thaw cycle. If you would like other spiking controls (refer to Control Table on our web site) please contact us by email. 3. Preparation of Total RNA (using TRIzol Reagent-GIBCOBRL) (Modified from Manufacturers instructions--see product insert for details or consult 'Protocols' at http://search.lifetech.com/) Different procedures can be adopted for making total RNA from different tissue types, like RNAwize from Ambion etc. 1. Homogenize 100-500 mg of tissue in liquid nitrogen. Add the frozen tissue to a prechilled mortar and pestle. Add enough liquid nitrogen to cover tissue. As liquid nitrogen evaporates (and for 1 minute thereafter) grind the tissue to a fine powder. 2. Add 1 ml TRIZOL per 100 mg of tissue and continue to homogenize. Sample may freeze at this point--continue grinding 1 minute after sample has thawed. 3. Transfer to RNase free centrifuge tubes. 4. Incubate 5 minutes at room temperature. 5. Add 0.2 ml Chloroform for 1 ml of TRIZOL and vortex 15 seconds. 6. Incubate 3 minutes at room temperature. 7. Centrifuge at 12,000 x g for 10 minutes. 8. Transfer the aqueous phase to a fresh RNase-free tube. 9. Precipitate by adding 0.5 ml isopropyl alcohol. Mix well by inverting, Incubate 10 minutes. 10. Centrifuge at 12,000 x g for 10 minutes. A very small pellet should be visible at the bottom of the tube 11. Discard supernatant by decanting. Quick spin. Remove remaining supernatant with pipetter. 12. Wash pellet by add 0.5 ml 75% ethanol (made with RNase-free water). 13. Centrifuge at 75,000 x g for 10 minutes. 14. Discard supernatant by decanting. Quick-spin in microfuge, if necessary. Invert the tube on a Kimwipe to draw off residual liquid. 15. Allow the pellet to air-dry for 5-10 minutes (e.g. until all of the ethanol has disappeared from the walls of the tube). DO NOT ALLOW THE PELLET TO FULLY DRY OUT. 16. Add 10 µl of RNase-free water and resuspend by pipetting 20-30 times. If necessary, heat at 65 °C for 10 minutes to dissolve RNA. 17. Quantify RNA using UV spectrophotometer and TE pH 8.0 as a buffer. Yield may also be quantified fluorometrically, e.g. using RiboGreen (Molecular Probes). 18. Check integrity of RNA sample and inspect for contamination with DNA by running 0.5-1.0 µg RNA with and without DNaseI treatment in a 2% agarose TBE gel (no need to run a denaturing gel). 19. Store at -20 °C for overnight or -80 °C for a longer term.
4. Preparation of polyA+ mRNA (using Dynabeads Oligo (dT)25-Dynal) (Modified from Manufacturers instructions--see product insert for details).1. Transfer 75 µg of RNA to an RNase-free tube. Adjust volume to 100 µl with distilled DEPC treated water. If the total RNA has already been diluted more than 100 µl then add the same total volume of 2X Binding buffer (solution B) to the Dynabeads to obtain a 1X Binding buffer concentration in the resuspension step below. 2. Heat RNA to 65 °C for 2 minutes. 3. In the meantime, resuspend the Dynabeads by shaking the vial gently to obtain a homogeneous dispersion of beads. Transfer 200 µl of resuspended Dynabeads from the stock tube to an RNase-free tube. Put the tube in the Dynal MPC (Magnetic Particle Concentrator-a magnetic device). After 30 seconds (or when suspension is clear) remove the supernatant. 4. Remove vial from Dynal MPC and wash once by resuspending in 100 µl of 2X Binding Buffer. Again place vial in the Dynal MPC and wait 30 seconds before removing buffer. 5. Transfer the vial to a rack and resuspend the beads in 100 µl 2X Binding buffer. Add more 2X Binding buffer, if necessary, as described in step 1. 6. Add the total RNA to the Dynabeads suspension. Mix gently but thoroughly and anneal by gently inverting vial for 3-5 min at room temperature. 7. Place the vial in the Dynal MPC for 30 seconds and remove the supernatant. 8. Transfer the vial to a rack and wash twice with 200 µl Washing buffer (solution E) using the Dynal MPC. The Dynabeads must be mixed thoroughly in the washing buffer and the supernatant completely removed between each washing step. 9. Elute the mRNA from the Dynabeads by adding 10-20 µl of Elution solution and keep at 65 °C for 2 min. Immediately place the tube in the Dynal MPC and transfer the supernatant containing the mRNA to a new RNase-free tube. You can reuse the Dynabeads up to 4 times for the same RNA sample. 10. Quantify RNA using UV spectrophotometer and TE pH 8.0 as a buffer according to standard protocol (see insert). Check quality of RNA sample by running 0.5-1.0 µg RNA in a 2% agarose TBE gel (no need to run a denaturing gel). 11. If the eluted mRNA is not to be used immediately, store at -70 °C. 12. Regenerate the Dynabeads for reuse according to manufacturers recommendations up to 4 times.
5. DIRECT LABELING OF PolyA+ mRNA 1. Combine in a 0.2 ml tube:
2. Incubate at 65°C for 10min. Note dyes are light sensitive and so tubes should be covered with foil to shield from light. 3. Transfer to ice and add:
4. Incubate at 42 °C for 90min 5. Add (IN THIS ORDER): EDTA (0.5M) 5 µl NaOH (1M) 10 µl 7. Incubate at 65 °C for 10min., 8. Cool to room temperature and add: Tris-HCl (1M , pH 8.0) 12.5 µl TE 400 µl 9. Apply to a Microcon-YM30 column and spin at 12,000 x g for 20 min, Wash column 4 times with 400 &181;l TE and spin 20 min. 10. Add 20 µl TE, mix well with pipette and elute by inverting the column and spinning at 12,000 x g for 5 min. 11. Use immediately or store in an opaque container at -20 °C for no longer than ~ 1 month. 12. You can test for label incorporation by diluting 1 ul of the product 10 100 times with TE and spotting 0.2 &181;l on a microscope slide. After drying, slide can be scanned at same laser power and PMT for both the channel. Quantify the spots and estimate the efficiency of incorporation for cy3 and cy5. Reagents and Materials DNTPs (Sigma DNTP100A-1KT) Cy3~, Cy5~dUTP (Amersham Pharmacia PA53022, PA55022) SuperscriptII (GIBCO-BRL) Microcon-YM30 column (Millipore 42410) 6. HYBRIDIZATION OF LABELED TARGETS TO MICROARRAYS 1. Prepare a hybridization chamber by cutting a paper towel in half, then folding the paper towel to fit along the long axis of a of a 50mL screw-top tube. Wet the paper towel with 2X SSC, then drain the excess liquid and seal the tube. Pre-heat the tube to 60-65 °C (you can loosen lid and microwave 10 sec and then place at 65 °C until you are ready to use it). 2. Combine Cy3 and Cy5 labeled targets. 3. Prepare the hybridization mixture in a 1.5 ml microfuge tube as follows (A=for 22x22 mm coverslip; B=for 22x40 mm coverslip):
Final volume is determined by practicing with a glass slide, reaction mix without labeled targets, the pipetters to be used, and a hybrislip on a 65 °C heat block. The goal is to have complete coverage without having excess solution flow outside the coverslip (Sigma Hybrislip Z36 591-2). I typically use 25 and 50 µl for the small and large hybridizations, respectively. 4. Close the tube and apply a lid lock. Denature at 95 °C for 2min. 5. Transfer to ice, then spin down briefly in a microcentrifuge. 6. Preheat a blocked and denatured slide on a 65 °C heat block. 7. Apply the hybridization mixture to the array, and quickly cover the liquid with a hybrislip (coverslips for hybridization, Sigma Hybrislips Z36,591-2). Immediately, transfer the slide to the pre-warmed 50 mL hybridization chamber (step 1). 8. Incubate overnight (8-12 hrs) at 60-65°C. If background is consistently high you can reduce hybridization time to 8 hrs. 9. Wash the array with gentle shaking using the following three wash steps: 1. 2X SSC, 0.5%SDS 55 °C, 5 min. (Coverslips should come off easily in this first wash) 2. 0.5X SSC room temp, 5min. 3. 0.05X SSC room temp, 5min. use clean slide staining chambers (Sigma S6141) for each rinse. I put the slides in a slide rack and transfer them (in the same rack) from rinse to rinse. NOTE: Make sure the slides are completely covered by rinse solution while shaking and never let the slides dry-if you must delay between steps, leave the slides in the rinse step they are currently in until you can proceed. If the slides dry you will have high background. 10. Immediately spin dry at low speed in a centrifuge (50-100 x g for 2-5 min) to avoid streaking. 11. Scan immediately. Last updated: Aug. 13, 2002 Return to Documentation Index | Return to Maize Gene Discovery Project index | Return to Homepage |
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