This protocol was optimized for a 20K array. The amount of RNA should be increased for a 38K array.
Oligo-oarray ProtocolStephan Eberhard, 02/07/2005
ALL solutions filtered (0.2 µm Nalgene Bottle-Top filter) and autoclaved (including ddH2O and milliQ water).
Immobilization
UltraGAPS Oligo-Slides:
1. Place arrays in humidity chamber filled with 80 mL ddH2O for 5 min.
2. Snap dry the arrays (array up) on 100ºC hot plate for about 3 sec.
3. UV-cross-link (UV Stratalinker) the oligos to the arrays at 600 mJoules (= 6000 x 100 µJoules).
You can keep the arrays in the slide holder until ready for prehybridization (after labeling the cDNAs).
Cleaning of total RNA
DNAse digestion
1. Start with <87.5 µl (no more than 45ug total RNA) of crude RNA preparation
2. Add 10 µl of Buffer RDD (Qiagen Rnase-Free Dnase Set, cat #79254) and 2.5 µl of DNase I stock solution.
3. Make the volume up to 100 µl with RNase-free water.
4. Incubate at RT for 10 min. Proceed with cleanup :
RNA cleanup using the Qiagen RNeasy MinElute Kit
This kit gets rid of the degraded DNA, DNAse, other proteins, tRNA, 5.5 rRNA, and other potential inhibitors of RT.
1. Add 350 µl of Buffer RLT (from the kit) to the 100 µl of RNA and mix thoroughly by pipetting.
2. Add 250 µl of 100% EtOH and mix thoroughly by pipetting.
3. Immediately load sample (700 µl) to an RNeasy MinElute column in a 2 ml collection tube.
4. Centrifuge at max speed for 15 sec.
5. Discard the flow-through and transfer spin column to a new 2 ml collection tube.
6. Add 500 µl of Buffer RPE (from the kit) and centrifuge at max speed for 15 sec.
7. Discard flow-through and reuse the same 2 ml collection tube.
8. Add 500 µl of 80% EtOH to the column and centrifuge at max speed for 3 min.
9. Discard flow-through and collection tube.
10. Transfer column to a new 2 ml collection tube and centrifuge at full speed for 5 min to get rid of remaining EtOH.
11. Discard flow-through. To elute the RNA, transfer the spin column to a new 1.5 ml Eppendorf. Place 20 µl of milliQ water[1] (preheated to 40ºC) onto the center of the column gel bed.
12. Centrifuge at max speed for 4 min.
13. Measure the OD260 of the eluted RNA and use 4 µg of this RNA for the subsequent labeling reactions.
Labelling reaction
1. Adjust 4 µg of cleaned RNA to 4 µl.
2. Add 1 µl of oligo-dT-(V), (T20VN);(2 µg/µl)
3. Heat 10 min at 70ºC and quickly chill on ice.
4. Add the following master mix:
5X Superscript Buffer:2 µl
0.1M DTT:1 µl
50X dNTPs[2]:0.2 µl
Cy3 or Cy5:1 µl
Superscript RT (200 U/µl):0.8 µl
(Final volume of reaction: 10 µl)
5. Incubate at 42ºC for 2h. Add 0.5 µl Superscript RT and incubate another 30 min.
6. Add 0.5 µl of 500 mM EDTA to stop the reaction.
7. Add 0.5 µl of 500 mM NaOH, incubate at 70ºC for 10 min to degrade RNA.
8. Add 0.5 µl of 500 mM HCl to neutralize the reaction.
Purification of labelled cDNAs using the Qiagen Qiaquick PCR purification kit
1. Combine the Cy3 and Cy5 and add 90 µl of DNase-free water.
2. Add 500 µl of Buffer PB.
3. Mix thoroughly by pipetting and add immediately to QiaQuick column.
4. Centrifuge at max speed for 1 min.
5. Discard the flow-through and reinsert the column in the same tube.
6. Wash with 750 µl of Buffer PE, centrifuge at max speed for 1 min, discard flow-through.
7. Repeat step 5.
8. Centrifuge the column at max speed for 2.5 min to elute any remaining buffer PE.
9. Transfer the column to a new 1.5 ml eppendorf tube.
10. Apply 50 µl of milliQ water preheated to 40ºC and let sit for 1 min.
11. Centrifuge at max speed for 4 min to elute the cleaned labelled cDNAs.
12. Dry down the samples in a SpeedVac (takes more than an hour). Cover SpeedVac with foil to avoid photobleaching of the probes.
During the drying time proceed to prehybridization of the slides:
Prehybridization
Prehybridization should be done immediately before hybridization.
1. Incubate slides in prewarmed Prehybridization Solution[3] at 42ºC for one hour.
2. Transfer arrays to 0.1X SSC and gently agitate at room temperature for 5 min.
3. Repeat step 3 in a fresh bath of 0.1X SSC.
4. Transfer arrays to ddH20 for 30 sec.
5. Dry arrays by spinning for 10 min.
Hybridization
1. Resuspend dried cDNAs in 25 µl of milliQ water preheated to 40ºC.
2. Prepare 2X Hybridisation Buffer, keep at 40ºC all the time, to avoid SDS precipitation.
3. Add 25 µl of 2X Hybridisation Buffer to each sample and resuspend thoroughly by pipetting.
4. Boil the samples for 3 min.
5. Meanwhile place arrays on kimwipe on plane surface. Prepare the Fisherbrand Large Coverslips (withour spacers).
6. Spin down the samples and pipett the probe onto the middle of the array. Carefully place Fisherbrand Large Coverslip onto the array. Be sure probe is evenly distributed all along the array.
7. Add 3 drops of 10 µl 3X SSCto the bottom of the array. Seal the chamber and incubate in 42ºC waterbath for 24 hours.
Post-Hybridization Washes
1. Preheat two 350 ml jars containing 2X SSC, 0.1% SDS to 42ºC at least 30 min (best to put the jars filled with buffer in the hot water bath).
2. Just before use, add 800 µl of freshly prepared 1M DTT to 800 mL of each washing solution.
3. Take out the jars. Disassemble the hybridization chamber.
4. Take out each slide individually and immediately hold with light dipping in the first preheated 2X SSC, 0.1% SDS jar, until coverslips freely move away from the slides.
5. Transfer slides to a new preheated jar containing 2X SSC, 0.2% SDS and dip gently for 5 min.
6. Transfer slides to 0.1X SSC, 0.1% SDS at room temperature and wash for 5 min.
7. Transfer arrays to 0.1X SSC and wash for 5 min.
8. Rinse arrays in 0.01X SSC for 10 sec and immediately spin-dry for 5 min.
9. Scan the arrays.
Solutions Required for Microarray Exepriments :
- Filtered and autoclaved ddH2O and milliQ water
ALL solutions to be filtered (Nalgene filter) and autoclaved. If prepared in this order, the same filter may be used for those solutions. Add 1.23 g of DTT to all washing solutions (10 mM final).
0.01X SSC :
20X SSC :400 µl
ddH2O :799.6 ml
2 Bottles of 0.1X SSC :
20X SSC :4 ml(x2)
ddH2O :796 ml(x2)
0.1X SSC, 0.1% SDS :
20X SSC :4 ml
10% SDS :8 ml
ddH2O :788 ml
2X SSC, 0.1% SSC :
20X SSC :80 ml
10% SDS :8 ml
ddH2O :712 ml
Use a new filter for the
Prehybridisation Solution (5X SSC, 25% formamide, 0.1% SDS, 0.1 mg/ml BSA)
20X SSC :200 ml
10% SDS :8 ml
BSA :80 mg
ddH2O :392 ml
Filter and autoclave. Only after that, add 200 mL formamide.
2X Hybridisation solution (6X SSC, 0.2% SDS, 0.4µg/µg poly(A), 0.4 µg/µl yeast tRNA)
For 200 µl :
20X SSC10% SDS4µg/µl Poly(A)tRNA (2 µg/µl)Formamide
60 µl4 µl20 µl40 µl76 µl
Microarray Protocol – Stephan Eberhard – 05/11/20041
[1] milliQ water has pH closer to 7.5 than ddH20 and pH is important for proper elution of the RNA.
[2] 50X dNTPs = 25 mM each, except for dTTP at 10 mM)
[3] See composition and preparation of all solutions at the end of the protocol.