cDNA Microarray Hybridization Protocol

Medina Lab cDNA microarray hybridization protocol – starting from aRNA

Prepared by: Mickey DeSalvo – 18 March 2008; modified by Chris Voolstra 7 April 2008; modified by Mickey DeSalvo 14 May 2009; modified by Mickey DeSalvo 14 January 2010.

 

  1. Turn on 63˚C water bath – monitor throughout the day to make sure it is at exactly 63˚.
  2. Reverse transcription and aa-dUTP incorporation
  1. Priming

i.     To 3ug aRNA, add 3uL pdN15 (use PCR tubes).

1.     Or 2uL of pdN9 (5ug/uL).

2.     pdN15 = random pentadecamer.

3.     pdN9 = random nonamer.

4.     If you need to re-suspend a new tube of pdN15, use 350uL of water — Poly N (15-mer) 50 OD from Operon (Item # SP180-1) has a conc. of 31.7ug/OD, so total mass of 1585ug.  Re-suspending in 350uL water gives a final conc. of 4.5ug/uL.

ii.     Bring final volume to 19uL with water.

iii.     Mix well and incubate at 70oC for 10min in PCR machine.

1.     Program “70” in “Main” folder (MJ PCR machine).

iv.     Place on ice immediately and leave for at least 5min.

  1. 25x aa-dUTP mix – prepare during priming reaction.

i.     Adjust volumes below according to # of samples.

ii.     aa-dUTP from Ambion is @ 50mM (50uL total per tube).

iii.     Ratio of aa-dUTP: TTP can be optimized – table below is a 4:1 ratio.

1.     We never deviate from the 4:1 ratio.

iv.     The reagents are kept in “Microarray Reagents” box in -80 freezer (second shelf from top, third rack from left).

v.     There may be old aliquots of 25x aa-dUTP mix in the reagents box – use your own discretion in using aliquots older than 6 months to 1 year.

Reagent Conc. uL Final conc.
dATP 100mM 5 12.5mM
dGTP 100mM 5 12.5mM
dCTP 100mM 5 12.5mM
TTP 100mM 1 2.5mM
aa-dUTP 50mM 8 10mM
water 16
     Total 40
  1. RT master mix (1x)

i.     Reagents are in “Microarray” clear plastic box in -20 freezer.

Reagent uL
5x First Strand Buffer 6
0.1M DTT 3
25x aa-dUTP mix 1.2
SuperScript III RT (200U/uL) 0.8
                                       Total 11uL
  1. RT reaction

i.     Add 11uL RT master mix to each tube.

ii.     Mix well and incubate 10min at 25oC then 2hr at 50oC.

1.     Program “2” in “Main” folder (MJ PCR machine) contains both 25˚ and 50˚C incubations.

iii.     To save time, you can label tubes and prepare hyb chambers (see step 6) during the 2hr reverse transcription.

  1. Hydrolysis

i.     Add 10uL 0.5M EDTA

ii.     Add 10uL 1M NaOH

iii.     Mix well and incubate at 65oC for 15min in PCR machine.

1.     Program “3” in “Main” folder (MJ PCR machine)

iv.     Add 50uL 1M HEPES pH 7.0 to each tube and mix well

  1. In preparation for dye coupling, take CyDyes out of freezer (also in “Microarray” clear plastic box in -20 freezer).

i.     Allow them to warm to room temp for 30min.

  1. cDNA purification
  1. MinElute columns are stored on bottom shelf of refrigerator.  Allow them to warm to room temp for at least 30 minutes.
  2. Remove RT reactions from PCR tube to 1.5 ml microfuge tubes.
  3. Add 300 μl Buffer ERC to tubes.
  4. Load samples into a MinElute Columns.
  5. Centrifuge at 13K rpm for 1 minute.
  6. Discard flow-through.
  7. Add 750 μl of Buffer PE to each column and spin at 13K rpm for 1 minute.
  8. Discard flow-through and spin again at 13K for 1 minute.
  9. Add 11.5 μl water to column matrix, let stand for 1 minute, and elute by spinning at 13K for 1 minute into a fresh 1.5 ml tube.
  10. Transfer 9uL to another set of tubes – set aside for dye coupling.
  11. Use the remaining volume (~1.5uL) for NanoDrop’ing.

Check cDNA synthesis on NanoDrop

  1. Choose “Nucleic acid” at main menu.
  2. Make sure to change constant to “33”.
  3. Use 1.5uL of your sample.

Dye Coupling

  1. Add 1uL 1M sodium bicarbonate pH 9.0 to the 9uL aliquots.

i.     If bicarb solution is older than 1 month, use pH paper to ensure that pH is still 9.0.

ii.     If you need to make new bicarb here is the recipe:

1.     Add 4.2g sodium bicarbonate to a 50 or 100mL beaker

2.     Add 40mL dH2O.

3.     pH solution to 9.0 with NaOH – bicarbonate will dissolve as the pH gets closer to 9.0

4.     Adjust volume to 50mL.

5.     Filter solution through 0.22um syringe filter.

  1. Spin tubes quickly.
  2. Spin CyDye tubes for 15sec at max speed.
  3. Re-suspend CyDyes in 18uL DMSO and mix by flicking the tube.

i.     DMSO aliquots are kept at -20 (box labeled “DMSO 1mL aliquots” on second shelf from top, second rack from left).

ii.     This will allow for 8 couplings with each dye.

iii.     You can re-suspend the CyDyes in a lower volume if you have less samples.

iv.     If you have more than 8 couplings, then re-suspend another tube of dye in the same volume of DMSO as the first.

  1. Spin re-suspended CyDye tubes.
  2. Add 2uL of Cy3 or Cy5 to the appropriate cDNA in bicarbonate buffer.
  3. Spin tubes quickly, mix by flicking, and spin quickly again.
  4. Incubate at RT in the dark for at least 1hr.

i.     While dye is coupling you can prepare hyb chambers if you haven’t already (see below).

  1. Prepare hybridization chambers:
  1. Open hyb chambers and clean with EtOH and Kimwipe.
  2. Place microarray face-up in hyb chamber.
  3. Label each microarray.
  4. Clean lifter-slip with EtOH and Kimwipe.
  5. Try to leave no pieces of dust or lint on lifter-slip.
  6. Place lifter-slip over the microarray (etchings bound the array).

i.     Make sure white strips are on the under side of the slip.

ii.     The white strips should be parallel with the long side of the slide.

  1. Using a pipet tip, position the lifter-slip directly over the microarray.

Purification of dye-labeled cDNA

  1. Add 300 μl Buffer ERC to tubes containing dye-coupling reaction.
  2. Load samples into a MinElute Columns.
  3. Centrifuge at 13K rpm for 1 minute.
  4. Discard flow-through.
  5. Add 750 μl of Buffer PE to each column and spin at 13K rpm for 1 minute.
  6. Discard flow-through and spin again at 13K for 1 minute.
  7. Add 14.5 μl water to column matrix, let stand for 1 minute, and elute by spinning at 13K for 1 minute into a fresh 1.5 ml tube.

Check dye coupling on NanoDrop

  1. Choose “Microarray” at the main menu.
  2. Use 1.5uL of your sample.

Hybridization

  1. Mix 12uL of Cy5-cDNA to 12uL of Cy3-cDNA in a PCR tube.
  2. Read steps C through F before proceeding.
  3. Add 10.5uL 20x SSC to each tube.
  4. Add 1.75uL 1M HEPES pH 7.0 to each tube.
  5. Add 32uL of dH2O.
  6. Tubes can be stored in the dark at 4oC for up to 24 hours at this point.
  7. When ready to proceed, add 1.75uL 10% SDS to each tube.

i.     If you are not going to store the tubes at 4˚C, then you can prepare a master mix of hyb. reagents (20x SSC, 1M HEPES, water, and 10% SDS) and add 46uL to each tube (24uL dye-labeled cDNAs + 46uL hyb solution = 70uL final volume).

  1. Mix well and incubate at 99oC for 2min in PCR machine.

i.     Do not overly tighten PCR lid – tubes can deform at 99oC.

  1. Allow tubes to cool at RT for at least 5min.
  2. Add 100uL 3x SSC to the wells at each end of the hyb chamber.

i.     The SSC will get wicked under the slide – this is OK.

ii.     Go back and fill each well with 3x SSC.

  1. Apply 70uL hyb solution under lifter-slip.

i.     Pipet slowly to avoid air bubbles.

  1. Close hyb chamber and incubate O/N at 63oC (water bath).

i.     Be very careful when handling hyb chamber – any drastic movements will cause hyb solution to be pulled under the microarray.

ii.     Wear 2 pairs of gloves when submerging chamber into water bath.

iii.     The hybridization should not exceed 16 hours.

  1. Microarray scanning preparation (the following day)
  1. Turn on NoZone filter.
  2. Make sure computer is turned OFF.
  3. Power ON the scanner.
  4. After scanner is ON, power up the computer.
  5. Start GenePix software.
  6. Let scanner warm up for 30min prior to scanning arrays.

Microarray washing

  1. All stock solutions and water should be FILTERED!
  2. How many arrays do you have?  You will need 2 50mL tubes filled with wash solution 1 and 2 50mL tubes filled with wash solution 2 for each microarray.

i.     Below is an example if 6 arrays are being washed.

ii.     If you have more than 6 arrays, then you’ll need to scale up the volumes for the two wash solutions.

  1. Prepare Solutions 1 and 2

i.     Solution 1 = 680mL water + 20mL 20x SSC + 1mL 10% SDS (0.6xSSC, 0.01%SDS)

1.     Fill 12 50mL tubes with Solution 1.

ii.     Solution 2 = 700mL water + 2mL 20x SSC (0.06xSSC).

1.     Fill 12 50mL tubes with Solution 2.

  1. Remove hybridizations from water bath.

i.     Wear 2 pairs of gloves!

  1. One at a time, submerge the array in the first tube containing Solution 1.

i.     Lifter-slip should fall off.

  1. Immediately transfer array to second tube containing Solution 1.
  2. Once all arrays are transferred to wash 1, cap all tubes, and invert for 1min.
  3. One at a time, transfer the arrays to the first tube containing Solution 2.
  4. Once all arrays are transferred to wash 2, cap all tubes, and invert for 1min.
  5. One at a time, transfer the arrays to the second tube containing Solution 2.

i.     Arrays can stay in this solution until dried.

  1. Dry one slide using the PicoFuge inside the NoZone workspace.

i.     Do not leave the NoZone door open for more than 30sec.  Keep the door closed as much as possible.

ii.     A 5sec spin is sufficient to dry the slide.

  1. Scan slide immediately, microarray face-down.

i.     Keep the remaining slides submerged in wash solution 2 while scanning other slides.

  1. After the first slide is scanned, dry another slide and scan it immediately.
  2. Keep dried slides in a box inside of the NoZone workspace.