1 Centre for Biomedical Research Department of Biology UNIVERSITY OF VICTORIA PO BOX 3020, STN CSC, VICTORIA, BC, CANADA, V8W 3N5 TELEPHONE: (250) FAX: (250) Page 1 of 5 KOOP LAB Version 3 Updated: Aug 2006 Aminoallyl Indirect cdna labeling System: Dual channel experiment (This protocol uses Invitrogen s SuperScript Indirect cdna labeling system Cat# L or L ) 1. First Strand cdna synthesis reaction: Set up a reaction tube for each sample to be labeled with Cy3 and a reaction tube for the sample to be labeled with Cy5. 2. Hydrolysis and Neutralization: 3. Purify First Strand cdna: Typically we precipitate the DNA in ethanol overnight 4. Labeling with Fluorescent Dye: Use: Cy3 Mono-Reactive Dye Pack (Amersham Biosciences, PA 23001) Cy5 Mono-Reactive Dye Pack (Amersham Biosciences, PA 25001) Make dyes fresh by adding 45 µl of DMSO supplied in kit to each vial. Mix well. Keep at -20 o C in a dark place until used. Add 5 µl of appropriate dye/dmso solution to cdna tube 6. Purify Labeled cdna: After eluting labeled cdna with 50 µl l of nuclease free water from S.N.A.P. columns, pool the Cy3 and Cy5 sample pair into one tube. Add 10 µl of 3M NaOAc ph 5.2, mix Add 300 µl of 95% ethanol, mix by inversion. Place in -20 o C for 1 hour or overnight. Centrifuge at 16,000 x g at 4 o C for 30 minutes. Remove ethanol
2 Page 2 of 5 Add 500 µl of 70% ethanol, mix with inversion, centrifuge at 16,000 x g at 4 o C for 10 minutes. Remove ethanol and let air dry for 10 minutes Rehydrate pellet with 26 µl of nuclease free water 7. Slide post-print processing: (Perform this just before prehybridization of microarray) Currently, the cdna slides are air-dried and crosslinked (120mJ) after printing. (This is the state at which they are shipped). Wash 2 times in 0.2% SDS for 5 minutes Wash 5 times in Milli-Q H 2 0 for 1 minute Dry slides by centrifugation 514 x g for 5 minutes Important: We have removed the boiling (denaturation) step as it was causing cdna to become unbound to the arrays resulting in comet streaks and high background. If you think your signal is low you may want to incorporate the boiling step back into the post processing protocol (see below). If comet artifacts appear on the array then do not continue with the denaturation step. Wash 2 times in 0.2% SDS for 5 minutes Wash 5 times in Milli-Q H 2 0 for 1 minute Place into boiling Milli-Q H 2 0 for 3 minutes Dry slides by centrifugation 514 x g for 5 minutes 8. Microarray Prehybridization: (helps reduce background) Incubate microarray for 90 minutes in 5XSSC, 0.1% SDS, 3% BSA (Fraction V) at 49 o C. Wash 3 time in Milli-Q H 2 0 for 20 seconds Dry slides immediately by centrifugation 514 x g for 5 minutes Place the arrays into 49 o C hybridization oven until cdna hybridization 9. Hybridizing of labeled cdna to microarray Thaw 2X Formamide buffer (50% Formamide, 8X SSC, 1% SDS, 4X Denhardt s Solution) in 55 o C water bath for 10 minutes. Ensure components are thoroughly mixed by inversion. Add appropriate reagents for 60 µl hybridization mixture: this volume works adequately with a 22x60 mm coverslip. Component Volume Concentrated cdna + H µl 2X formamide hybridization buffer 30 µl LNA dt blocker (Genisphere Cat# CW3910 or CW3920) 4 µl OPTIONAL: Cot DNA or other blocking DNA may be added in this mix. Denature Cot DNA prior to adding (i.e. 95 o C 10 min.)
3 Page 3 of 5 Incubate hybridization mixture at 80 o C for 10 minutes then hold at 65 o C until loaded onto array. Add 60 µl of hybridization mixture (avoid any precipitate if present) to array & place coverslip (22 x 60 mm HybriSlip) over array. Place array into hybridization chamber. Hybridization chambers should be washed and free of lint. Use Milli-Q water and air dry. Depending on style of hybridization chamber, either fill reservoirs at ends of chamber with 13 µl of nuclease free water, or saturate a small piece of Whatman paper with water and add to reservoirs at ends of chamber. Incubate for 16 hours (no longer) at 49 o C in a humidified chamber. Methods for adding hybridization mix to the array: 1. Add hybridization mixture directly to array, taking care not to touch the array with the pipette tip. Deposit it in a small pool or bead at the top of the array. Hold coverslip by its edges and touch it down, at an angle to the deposited hybridization solution. Slowly lower the coverslip to the array. 2. Add the hybridization mixture to the coverslip and gently, but smoothly, flip the coverslip onto the array, again touching down at an angle and then gently lowering the coverslip to the array. 10. Post cdna Hybridization Wash These wash conditions may need to be optimized on a per lab basis. Use reagent grade water (i.e. MilliQ) when making up wash buffers and filtersterilize all stock solutions (autoclave 20X SSC stock solution). Be aware that MilliQ water filtration systems may have some oxidative agents that degrade Cy5. Some people add a reducing agent to MilliQ water in preparing the first 2 wash buffers. (i.e. final concentration of mm DTT). Use fresh DTT as old DTT can cause hazing in the Cy3 channel. Important: Perform all washes in the dark to minimize degradation of the Cyanine dyes Float off coverslip in 2X SSC/ 0.1% SDS heated to 49 o C. Do not force coverslip off. Wash component Duration Frequency 2X SSC/ 0.1% SDS heated to 49 o C 10 min 1X 2X SSC/ 0.1% SDS Room temperature 5 min 2X 1X SSC Room temperature 5 min 2X 0.1X SSC Room temperature 5 min 2X Wash each slide individually in 50 ml conical tubes, with agitation, or wash in 400 ml glass slide dish, in metal slide holders either on a shaker, or with a small, clean stir bean on a magnetic stir plate. Do not wash more than 4 slides per slide dish.
4 Page 4 of 5 Immediately after final 0.1X SSC wash, transfer array to 50ml centrifuge tube with a kimwipe at bottom to absorb extra moisture and centrifuge 514 x g for 5 minutes. Orient slide so array surface is facing out in the centrifuge, label is down. Slides can also be centrifuged in slide tray holders. Place slides in the dark until ready to scan. Signal will decrease overtime so it is best to scan as soon as possible
5 Page 5 of 5 Materials/Reagents Vendor Catalogue Number SuperScript Indirect cdna labeling system (10 assays) SuperScript Indirect cdna labeling system (30 assays) Invitrogen L Invitrogen L LNA dt blocker (10 assays) Genisphere CW3910 LNA dt blocker (20 assays) Genisphere CW3920 Cy3 Mono-Reactive Dye Pack (5 vials) Cy5 Mono-Reactive Dye Pack (5 vials) Amersham Biosciences Amersham Biosciences PA PA Hybridization chambers (5 chambers) Corning 2551 Hybri-slips (size: 22mm x 60mm) Sigma Aldrich Z370274