title: cDNA microarray Van Andel Institute for Day1 samples
status: Public on Aug 03 2006
contact.city: East Lansing
contact.first: Zheng
contact.last: Li
contact.email: lizheng1@msu.edu
contact.state: MI
contact.address: 2527 Engineering Building
contact.department: Chemical Engineering and Materials Science
contact.country: USA
contact.web_link: http://www.msu.edu/~lizheng1
contact.institute: Michigan State University
contact.phone: 517-432-1003
organism: Homo sapiens
manufacture_protocol: MicroArray Protocol For Total RNA
  Ramsi Haddad
  3/14/02
  This protocol requires 50 µg of each type of RNA to be compared for each microarray. The word probe will be used to describe the labeled cDNA that binds to the DNA on the microarray.
  Materials
  The following is a list of supplies required to perform cDNA microarray hybridizations. Most items are available in the Invitrogen Freezer Program.
  Company Cat. Number Description Amersham Pharmacia Biotech 27-7836-01 poly d(A) sodium salt or use Sigma poly d(A) Perkin Elmer NEL576 Cy3-dCTP NEL577 Cy5-dCTP Ambion 9915G DepC treated water 9480 7.5 M LiCl solution Brinkmann/Eppendorf 0032 005.330 Phase Lock Gel-Heavy, 50 mL tubes Qiagen 28104 QIAquick PCR purification Kit (50 columns) Roche 799-017 Rnasin (2000 U) 1467140 ss Salmon Sperm DNA PGC Scientifics 62-6504-04 22 X 40 mm hybrislip, pack of 100 Fisher 42416 Microcon-50 microconcentrators (Amicon no. 42416) 2551 CMT Hybridization Chamber (Corning no. 2551), 5 chambers 08-812 Histology staining dish (horizontal) 08-953-E Jewelers Microforceps, 10.8 cm (straight) 13-812-14 Kelly Forceps, curved, 140 mm 12-548-5J 24 X 40 mm glass coverslips, 1 oz pack 05-669-39 1.5 mL microfuge tubes with screw cap O-ring seal, 500 tubes Gibco/BRL Invitrogen 15279-011 Human Cot-1 DNA, 500 ug 18064-014 SuperScript II RT. 18440-016 Mouse Cot-1 DNA (if required) 10297-018 100 mM dNTP Set 15596-026 TRIzol Reagent, 100 mL (200 mL bottle is also available) 15634-017 Herring Sperm DNA, 5 X 1 mL or below 15632-011 Salmon Sperm DNA, 5 X 1 mL or above Promega PRM-4261 Rnase One Sigma D-2532 Denhardt's Solution 50X concentrate, 5 mL S-2015 20X SSPE, 1 L S-6639 20X SSC, 1 L F-9037 Formamide, 100 mL P-9403 Poly d(A) or use Pharmacia Amersham. R-7876 2500 U tRNA (type V wheat germ) Ambion 9906 10 X 50mL DepC treated water 9480 100 mL 7.5 M LiCl solution for RNA purification, 100 mL
  Reverse transcription of probes.
  1. Start with 50 µg RNA of each type in 9 µL water.
  2. Have the following temperatures available:
  a) 37°C, 50°C water baths
  b) 42°C, 68°C and 95°C heating blocks
  c) Thaw 5X 1st strand buffer, 0.1M DTT, oligo d(T)20VN, Cy3-dCTP,
  Cy5-dCTP, 10X Low dC/dNTP, microarray pre-hybridization and
  hybridization solution.
  3. Anneal oligo dT primers to RNA.
  a) Add 1 µL dTVN primer (0.5 µg/µL) and incubate 5 minutes at 68°C
  b) Give a quick spin to collect condensation. Keep at room temperature or
  solution will get too viscous.
  4. Prepare 2 separate tubes of each fluorescent mix.
  (X4.5) (X 5.5 reactions)
  a) 4 µL 5X 1st strand buffer 18 (22 µL)
  b) 2 µL 0.1 M DTT 9 (11 µL)
  c) 2 µL 10X low dC dNTP 9 (11 µL)
  d) 2 µL Cy3 or Cy5 dCTP 9 (11 µL)
  e) 0.5 µL RNAsin 2.2 (2.7 µL)
  f) This should give a total of 10.5 µL.
  5. Mix annealed RNA with appropriate fluorescent mix.
  6. Add 1 µL SuperScript II RT per RNA reaction and incubate at 42°C for 45 minutes.
  Using another pipettor at ~15 µL, pipette up and down to mix. This mixing step is VERY important. Since Cy5 and Cy3 are light sensitive, cover tops of tubes with foil during reaction. Do not let the temperature exceed 44°C.
  7. Add another 1 mL SuperScript II RT per RNA reaction, mix with 15 mL pipette, and
  incubate at 42°C for another 45 minutes. Subsequently place at 94°C for 2 minutes,
  quench on ice and then combine reactions in one tube.
  GO TO STEP 10 FOR MICROARRAY PREHYBRIDIZATION, THEN
  COMPLETE STEPS 8 AND 9.
  8. Clean up cDNA.
  a) Get rid of RNA
  i) Add 50 µL water
  ii) 10 µL 10X RNAse One buffer
  iii) 2 µL RNAse One
  iv) Incubate at 37°C for 30 minutes.
  v) This should give a total of 99 µL
  b) Remove unincorporated label and small cDNA fragments using the QIAquick PCR
  Purification Kit.
  i) Add 500 µL Qiagen Buffer PB to probe and mix.
  ii) Apply the sample to QIAquick spin column and centrifuge at Room
  Temperature 45 seconds, 10 000 g.
  iii) Discard flow through, add 750 µL Buffer PE (wash buffer) to spin column
  and centrifuge for 45 seconds, 10 000 g.
  iv) Discard flow through and spin again for an additional minute to remove
  residual Buffer PE.
  v) Elute probe by adding 50 µL Buffer EB (elution buffer) to the center of the QIAquick membrane, let sit one minute and then centrifuge for one minute.
  vi) Place probe eluted from QIAquick column on a MICRON YM 50 column and centrifuge at Room Temperature for 1.5 minutes, 12400g. This should concentrate the probe to less than 15 µL.
  vii) To collect purified probe, invert column into new tube. Set centrifuge to 3000 RPM and spin for 15 seconds.
  viii) Check the volume and bring up to 15 µL.
  9. Probe pre-hybridization.
  a) To purified probe, add 28.8 µL probe hybridization and 2.3_µL 20X Blocking solution to probe. This gives a total of 46 µL (extra is due to evaporation during prehybridizaiton ).
  b) Place in 94°C heating block for 2 minutes.
  c) To remove any particulate matter, centrifuge at 14K g for 5 minutes and transfer to a clean tube with a rubber o-ring cap to prevent excessive evaporation.
  d) Place probe at 50°C for one hour (minimum 45 minutes ) to pre-hybridize. Do not exceed 1.5 hours due to evaporation.
  e) Once probe has pre-hybridized, centrifuge 5 minutes at14K g and use 32-36 µL per microarray for hybridization in step 11.
  10. Prehybridize microarrays.
  a) Add 34 µL of microarray prehybridization buffer solution (frozen aliquots at -20°) over microarray. This is accomplished by slowly ejecting the solution to form a drop on the pipettor tip and gently touching the drop (NOT the tip) to the middle of the microarray.
  b) Place coverslip over the prehybe. Use Plastic Coverslips or you’ll have a difficult time removing it later! Coverslips are sandwiched between two plastic films. Break one film by bending coverslip to one side and remove coverslip with forceps. This gives a coverslip with the plastic film on one side. Using 90° forceps, lay so that tips are on line defining arrray. Coverslips will use this as a fulcrum to prevent sliding. While holding the corner of the coverslip, place the edge of the coverslip at the border of the micro array at ~45° angle, against the forcep. A line of prehybridization solution will form at the contact. Gently lower the slide until its almost down and then let go with the forceps. Do not drop the slide from too high a height or it will move. Once the coverslip is positioned, do NOT move it.
  c) Place the microarray with coverslip into hybridization chamber. Add
  10 µL of 50% formamide/50% water to each dimple at the end of the CMT hybridization chamber.
  d) Place cover on chamber and place in 50°C water bath. Allow prehybridization of microarray to proceed for at least two hours.
  11. Remove prehybridization and add hybridization solution.
  a) Rinse hybridization chamber and dry the outside of the chamber well. Make sure to keep chamber level at all times. Remove microarray from chamber and place on paper towel.
  b) Remove coverslip and microarray prehybridization solution. Using the left hand, tilt the slide so that its long edge remains in contact with paper towel and an acute angle forms between the coverslip side of the slide and the paper towel. Grasp the top corner of the coverslip with forceps and while holding coverslip in place, gently tilt the slide away. The prehybe solution will be seen to separate from the coverslip just like a acrlyamide gel pulling away from a glass plate. Make sure the slide and coverslip maintain contact at the bottom edge where all the solution will gather. Once the coverslip touches the paper towel, the prehybe will blot all over.
  c) Add 32-36 µL hybridization solution to each microarray and hybridize overnight. Add 10 µL of 50% formamide/50% water to each dimple at the end of the CMT hybridization chamber.
  The next day, wash and scan the microarrays and scan them.
  12. Prepare 500 mL of the following wash solutions:
  a) 1X SSC/0.1% SDS (25 mL 20X SSC/2.5 mL 20% SDS)
  b) 0.2X SSC/0.1% SDS (5 mL 20X SSC/2.5 mL 20% SDS)
  c) 0.2X SSC (5 mL 20X SSC)
  d) 0.1X SSC (2.5 mL 20X SSC)
  13. Rinse hybri