Mouse 15K microarray hybridization protocol

protocol by Dennis Wigle        last updated May 2002 (modified from Niki Dobric & the OCI array facility)

 NB: It is very important to order and prepare all reagents, and review recipes before beginning protocol
  check here

RNA Cleanup – Qiagen kits
We have been cleaning up RNA purified using phenol-based methods (GITC, Trizol, etc.) prior to probe preparation. This seems to produce more efficient and consistent labelling. This step is not necessary if you have used a column-based purification initially (eg Qiagen kits). We have successfully used the QIAEASY mini columns as an RNA clean-up step.

Probe Preparation (Amino-allyl Coupling)
Note on amount of total RNA used for microarray:
For human 1.7K chip use 5-10ug of total RNA in RT reaction.
For human 19K, mouse 7K, 14K, and 15K chip use 10-20ug of total RNA in RT reaction.
note:If your RNA supply is not limited always use more rather then less.

DAY 1:
In the first step amino allyl dUTP (AA-dUTP), an amine-modified nucleotide, is incorporated during reverse transcription. Subsequently, monofunctional forms of Cyanine 3 and Cyanine 5 dyes are reacted with the amine-modified cDNA. We find this gives better labelling efficiency with less input RNA and less dye bias in fluor-flips than incorporating Cy3 & Cy5-dCTP directly during reverse transcription.

RT reaction (SuperscriptII RNAseH- Reverse Transcriptase)
 Contents  conc of stock solution  amount (ul/tube)
first strand buffer 5x 8 ul
AncT mRNA primer 200 pmoles/ul 0.75
DTT 0.1M 4
total RNA 5-20 ug 21.6
TOTAL VOLUME   34.35
  • Incubate reactions at 65oC for 5 minutes
  • Incubate reactions at 42oC for 5 minutes
  • While at 42oC add the following mix to each tube 
 Contents  conc of stock solution  amount (ul/tube)
dG,dA,dC mix 20mM each 1
dTTP 5mM 1.3
aa-dUTP 10mM 1.35
SupersciptII 200 units/ul 2
TOTAL VOLUME   40
Incubate reactions at 42oC for 2 hours
Add 8 ul 1 M NaOH and heat at 65 C for 15 minutes to hydrolyze remaining RNA.
Add 8 ul 1 M HCl and 4 ml 1 M Tris-Cl, pH 7.5 to neutralize the solution

Probe Purification and Precipitation
At this point reactions are purified using the Qiagen PCR Purification Kit (or mini-prep kit; both work). All traces of Tris must be removed to prevent reaction of the amine groups on Tris with the monofunctional NHS-ester of the Cyanine dye.

  1. Add 38µl dH2O to bring each reaction volume to 100 µl.
  2. Add 500µl PB buffer to each 100 ul reaction and mix.
  3. Apply solution to the column included with the kit and spin at 10,000 rpm for 1 min. Re-apply eluant to column a second time and spin again. Discard flow-through
  4. Wash with 600µl 75 % EtOH and spin at 10,000 rpm for 1 min. Discard flow-through and repeat this wash step once.
  5. Spin the column for one additional minute to ensure the membrane is dry
  6. Use 100µl dH2O to elute the cDNA. Sit for 5 minutes before spinning. Repeat this once
  7. Add 50µl 3 M sodium acetate.
  8. Add 500µl 100 % EtOH (alternatively, use isopropanol) and precipitate at -20 °C for 1 hr or longer. Spin at top speed for 20-30 minutes to ppt.
  9. Wash the pellet with 80 % EtOH. Make sure that all ethanol is removed, however do not allow the pellet to dry completely.
Labeling Reaction with Monofunctional Reactive Cyanine Dye

  1. Resuspend cDNA in 6µl dH2O
  2. Add 3µl 0.3 M sodium bicarbonate, pH 9.0 Please see note about aliquoting dye at the end of this protocol
  3. Add 1.0µl dye to the reaction and incubate in the dark at room temperature for 40 minutes to 1 hour.
DAY 2: Quenching
Before combining Cy3 and Cy5 samples for hybridizations the reactions must be quenched to prevent cross coupling.
  • Add 4.5ml of 4M hydroxylamine
  • Incubate reaction at RT for 15 minutes in dark (i.e. place in a drawer)
Note: Lately we have been skipping the quenching step. If you do this, it is important to purify and ppt the probes separately and only combine them prior to hybridization to prevent cross-coupling.

Purification of Fluorescently Labeled Probe Using Qiagen PCR Purification Kit

  1. Add 90µl dH2O to bring each reaction volume to 100 µl
  2. Add 500µl PB buffer to each 100 µl reaction and mix
  3. Apply solution to the column included with the kit and spin at 10,000 rpm for 1 min. Re-apply eluant to column a second time and spin again. Discard flow-through.
  4. Wash with 600µl 75 % EtOH and spin at 10,000 rpm for 1 min. Discard flow-through and repeat this wash step 2 times.
  5. Spin the column for one additional minute to ensure the membrane is dry.
  6. Use 100µl dH2O to elute the cDNA. Sit for 5 minutes before spinning. Repeat this once.
  7. Add 500µl 3 M sodium acetate.
  8. Add 500µl 100% EtOH (alternatively, use isopropanol) and precipitate at -20 C for 1 hr or longer. Spin at top speed for 20-30 minutes.
  9. Wash the pellet with 80 % EtOH. Make sure that all ethanol is removed, however do not allow the pellet to dry completely.
  10. Resuspend the pellet in 2.5µl dH2O.
  11. Prepare the hybridization solution as usual.
Hybridization:

Pre-hybridization:

  1. Make pre-hybridization solution by adding the following per 100ml of DIG Easy Hybridization solution: 5ml 10mg/ml tRNA + 5ml 10mg/ml Salmon Sperm DNA + 10ml of 10%BSA
  2. Set up hybridization chambers (We use empty pipette tip boxes – add some ddH2O to the bottom of each box to prevent drying )
  3. Add pre-hybridization solution in between two slides or under a coverslip... do one slide at a time and immediately put slide into hybridization chamber to prevent drying
  4. Carefully wrap the chamber with saran wrap to prevent drying
  5. Incubate slides at 37oC for 30 minutes to 2 hours
  6. Carefully remove coverslip or separate slides by dipping array in dH2O
  7. Quickly rinse slides by dipping in dH2O several times
  8. Quickly dip slides in isopropanol and immediately place on hard surface to dry

Note: Lately we have been skipping the pre-hyb step
I am not sure it makes a difference to the overall quality of the resulting hybridization on the array platforms we use here.

Hybridization:

  1. Make hybridization solution by adding the following mix to your probe: 5ml 10mg/ml tRNA + 5ml 10mg/ml Salmon Sperm DNA+100ml DIG Easy Hyb    (We typically use 125ml of hyb solution for the mouse arrays)
  2. Heat hybridization solution at 65oC for 2 minutes
  3. Cool and maintain at RT
  4. Add all of the mixture to slides as described in pre-hybridization (Do one slide at a time and immediately put slide into hybridization chamber to prevent drying)
  5. Carefully wrap the chamber with saran wrap to prevent drying
  6. Incubate slides at 37oC overnight (at least 12 hours)

DAY 3: Washing

  1. Remove hybridization chambers from the incubator
  2. Remove a slide pair or a single slide with a coverslip from the chamber and quickly remove coverslip or separate slides by dipping array in 0.1XSSC;0.1%SDS
  3. After slides are separated load slides into a slide holder immersed in 0.1XSSC;0.1%SDS
  4. Repeat for each slide pair, loading into immersed slide holder as soon as coverslip or another slide has been removed
  5. Wash slides 5 times 5 minutes each at room temperature in clean slide staining boxes containing 0.1XSSC;0.1%SDS with gentle agitation
  6. Wash slides 5 times 5 minutes each at room temperature in clean slide staining boxes containing 0.1XSSC with gentle agitation
  7. Place slides in black slide boxes with fresh Whatman paper on the bottom of the box
  8. Spin dry @ 800rpm for 10 minutes
  9. Scan arrays as soon as possible, although signal can be stable for up to 3 days
  10. Store slides in the dark

Scanning: PDF file on scanning can be downloaded here..........)
 

Notes on splitting up work across multiple days:
You can stop and freeze samples for extended time periods at the indicated juncture in the protocol. Lately I have been doing the RT, cleaning it up, and then ppt o/n for day 1; followed by labelling in am next day, clean up and ppt over lunch/early pm, then hyb o/n; followed by wash and scan on day 3. I find this splits things up evenly and also maximizes time for ppt (hence increasing yields).