Microarray protocol Emmanuela Marchi PhD Dept. Pharmacology UFIR - Comparative nutritional systems biology Focus Team Post-doc emanuela.marchi@unifi.it 16 April 2009 When citing this SOP you should acknowledge both NuGO and the appropriate NuGO partner institution that has made the SOP available. Please use a form of words such as: We used the NuGO Standard Operating Procedure (SOP) number 63 produced by the University of Florence. Details of the SOP are available via the web link: http://www.nugo.org/frames.asp?actionid=38873&action=loginfrompp Reagents and Materials Nuclease Free Water Ambion #4387936 Primer Solution Oligo(dT) 12-18 Primer Invitrogen #18418-012 Hexanucleotide Mix 10X ROCHE #11277081001 Combine equal volumes of each primer mix to obtain Primer Solution. Superscript II Reverse Transcriptase Kit Invitrogen # 18064-014 Kit content: Enzyme 200U/µl (10,000 U) 5X First-Strand Buffer 0.1M DTT Aminoallyl dutp (AA-dUTP) SIGMA #A0410 (sodium salt powder) Dissolve to 100mM with nuclease free water 50X Aminoallyl-dUTP/dNTP Solution Combine: 100mM datp 10µl Invitrogen #10297-018 P/N 55082 100mM dctp 10µl Invitrogen #10297-018 P/N 55083 100mM dgtp 10µl Invitrogen #10297-018 P/N 55084 100mM dttp 6µl Invitrogen #10297-018 P/N 55085 100mM AA-dUPT 4µl SIGMA #0410 (for more stock solution scale up all volumes) Sodium Hydroxide 1N solution (dissolve in nuclease free water) MERCK #1.06498 EDTA 0.5M, ph 8.0 Ambion #AM9261

HEPES 1M, ph 7.5 (dissolve in nuclease free water) SIGMA #H3375 Sodium Bicarbonate 1M, ph 9.0 (dissolve in nuclease free water) Cy3 Mono-Reactive Dye Pack Cy5 Mono-Reactive Dye Pack DMSO FLUKA # 41639 Amersham - GE Healthcare #PA23001 Amersham - GE Healthcare #PA25001 SSC Buffer Ambion #AM9763 20X 3X (add 45ml of 20X SSC to 255ml of nuclease free water, in a clean nuclease-free container) SDS 10% Solution Ambion #AM9822 PCR Purification Kit Qiagen #28106 Microcon YM-30 centrifugal filter devices Millipore # 42410 Ultrafree-MC 0.45µm centrifugal filter devices Thin-Wall PCR Tubes 0.5ml Slide Hybridization Chamber Lifter (cover) Slips Millipore #UFC30HVNB Ambion #AM12275 Telechem International Inc #AHC-1 Erie Scientific Company #22X50I-2-4711 Instruments Speed-vac Water bath at 65 C Microcentrifuge Thermocycler Scanner Pre-procedure - Prepare Cy3 e Cy5 aliquots: Work away from direct light sources. Resuspend the dye powder in 20 µl of DMSO. Aliquot 2 µl of the suspension in 10 nuclease free tubes,

appropriately labelled. Let the aliquots completely dry in a speed-vac. Generally it takes about 1 hour. Store the aliquots at 4 C and use within 2 months from preparation. - Prepare Primer Solution (see above) - Prepare 50X Aminoallyl-dUTP/dNTP Solution (see above)

Main procedure RNA microarray labeling procedure Aminoallyl dutp method 1. For each reaction, calculate the volume of sample needed to process 35-40 µg of total RNA. If this volume exceeds 14.5 µl, the RNA source sample will require concentrating. 2. Add samples to PCR tubes, and bring their volumes to 14.5ul with nuclease free water. Keep these tubes at on ice. 3. Add 2 µl of the Primer Solution to each tube. Cap, vortex, and briefly centrifuge each. 4. Place these reaction tubes into a thermocycler, and incubate at 70 C for 10 minutes. Put on ice for at least 5 minutes. Spin down. 5. During this incubation, prepare the following Reaction Solution (x1 ): 5x Buffer 6 µl 50x Amino Allyl-dUTP/dNTP 0.8 µl 0.1M DTT 3 µl DEPC Water 2 µl Total Volume 11.8µl 6. Once the above incubation is complete add 11.8 µl of the Reaction Solution to each tube. 7. Add 2 µl of Superscript II Reverse Transcriptase to each tube, and briefly vortex. 8. Place these tubes into a thermocycler, and incubate at 42 C for between two and three hours. After 1.5 hours add 1 µl of Superscript II Reverse Transcriptase to each tube. 9. After the 2-3 hour incubation, remove the tubes from the thermocycler and add 10 µl of 1N NaOH and 10 µl of 0.5M EDTA to each. 10. Vortex each tube and incubate for 15 minutes using the thermocycler at 65 C. 11. After this incubation, add 25 µl of 1M HEPES to each tube. * 12. For each sample, weigh and label a Microcon YM-30 filter, and assemble with its collection tube. 13. Add 450 µl of nuclease free water to each tube, and transfer samples to the appropriately labeled YM-30 filters unit. 14. Centrifuge these units at 10,000 x g for 8 minutes. 15. Remove the effluent from the collection tubes, and add 470 µl of nuclease water to the filter units. 16. Again, centrifuge for 10,000 x g for 8 minutes. 17. Remove the YM-30 filter units from their collection tubes and weigh in order to determine the retained liquid volume: 1mg = 1µl 18. Continue centrifuging these units at 10,000 x g until the volume is between 10 µl and 18 µl.

Use the following chart to estimate duration. > 100 µl 4 min 50-99 µl 2 min 25-49 µl 1 min < 25 µl Pulse to 10,000 x g, then stop Avoid centrifuging to dryness, as it will be impossible to recover the sample. 19. Invert the filter into a clean collection tube, and centrifuge for 1,900 x g for 5 minutes. 20. Adjust the volume to 10 µl (avoid to have more volume!!). * 21. Add 1 µl of the Sodium Bicarbonate solution to each collected sample. 22. Pipette each of these samples into an appropriate labeled Cy-dye tube. Vortex, and centrifuge to ensure that the liquid is at the bottom of the tube. 23. Incubate this dye-probe coupling reaction for at least 1 hour at room temperature in total darkness. Probe purification 24. Add 50 µl of nuclease free water to each reaction tube and 20 µl of Sodium Acetate 3M ph 5.5. 25. Add 500 µl of PB Buffer (from the PCR Purification Kit) to each tube. 26. Add these solutions to appropriately labeled Qiaquick columns (from the PCR Purification Kit). 27. Centrifuge at 18,300 x g for 1 minute. 28. Decant the effluent from the collection tubes, and observe the columns' glass-fiber filters. Successful probe synthesis will be indicated by obvious blue and pink tints to these filters. 29. Add 700 µl of PE Buffer to each column, centrifuge at 18,300 x g for 1 minute. Decant effluent. 30. Repeat step #29. 31. Centrifuge the columns again, empty, to ensure dryness. 32. Place the columns into fresh 1.5 ml collection tubes. 33. Add 30 µl of EB Buffer to the columns' glass-fiber surface, incubate at room temperature for 5 minutes and centrifuge at 18,300 x g for 1 minute. 34. Repeat step #33. * 35. Assemble one YM-30 filter unit for each dual-hybridization sample. Into each unit, combine the appropriate 60 µl of collected Cy3 and Cy5 labeled probes purified above. 36. Centrifuge the filter units at 10,000 x g for 1 30. 37. Invert the filter into a fresh collection tube, and centrifuge at 1,900 x g for 5 minutes. 38. Adjust the collected probe solution to 70 µl with nuclease free water. * *At this point you can suspend procedure and store samples at -20 C

39. Add the following reagents to each probe: Hybridization 20x SSC 1M HEPES, ph 7.5 8.4µl 1.34µl 40. For each combined probe, add 10 µl of nuclease free water to a 0.45 microfilter unit, and centrifuge it at 18,300 x g for 1 minute. 41. Discard the effluent and collection tubes, and add each filter unit to fresh 1.5 ml collection tubes. 42. Add a probe sample to each of these tubes, and centrifuge at 18,300 x g for 1 minute. 43. To each effluent-probe add 1.26 µl of 10% SDS. 44. Transfer each probe sample to individually labeled PCR tubes, vortex, and centrifuge briefly to ensure sample is at the bottom of these tubes. 45. Place tubes in thermocycler, denature 100 C for 2 minutes. Use the hot bonnet. 46. During this incubation, prepare the Slide Hybridization Chambers. Remove dust from the chambers using a stream of pressurized inert gas. Place microarray slides upright in these chambers. Select an appropriate size Lifter Slip, clean using pressurized gas, and place onto the appropriate area of the microarray, making sure the "lifters" are face-down on the slide. 47. Pipette 120 µl of 3X SSC into both of the chamber's hydration wells. 48. After the above incubation, pipette the probe samples onto the appropriate arrays. Place the pipette tip at the open interface of the Lifter Slip and the array, without touching either. Slowly pipette the sample, filling the entire area under the Lifter Slip. Pipette the entire sample, even if the sample starts to protrude from the sides of the slip. This will ensure against drying. If after pipetting, the probe sample does not fill the entire Lifter Slip area, carefully press down onto the slip with a pipette tip. 49. Secure the covers on the hybridization chambers. Slightly tighten the screws with a screwdriver. 50. Place the hybridization chambers into a 65 C water bath, and incubate overnight in total darkness.

Pre-procedure Post-Hybridization Slides Washing - Prepare the following solutions, and place them into tanks. Wash Solution 1: 387 ml Milli-Q Water 12 ml 20X SSC 1 ml 10% SDS Wash Solution 2: 798ml Milli-Q Water 2 ml 20X SSC - Prepare a slide rack with the appropriate number of balance slides, and place in centrifuge on rotor-platform. - Set centrifuge settings at 1500rpm, 2 min, and 25 C. Main procedure 51. Place a slide rack into the tank containing Wash Solution 1. 52. Remove the hybridization chambers from the water bath while keeping them level. 53. Remove each slide from the chambers using forceps. 54. Invert each slide within Wash Solution 1, and agitate until Lifter Slips fall off. 55. Repeat for each slide. 56. Place slides in rack within Wash Solution 2. 57. Plunge rack up and down 40 times within this solution. Wash with energy. 58. Quickly transfer rack to rotor-platform, and spin dry. 59. Scan slides as soon as possible.