Microarray Procedure

1.        Pour plates

2.        Spot/spread bacteria

3.        Clorox bleach worms and egg drop

4.        Nematode Plae Wash

5.        RNA extraction

6.        mRNA prep

7.        cDNA synthesis

Manual Hybridization

8.        Prehybridization of array

9.        Hybridization of cDNA

10.    Post cDNA Hybridization Washes 1-3

11.    Hybridization of 3DNA

12.    Post 3DNA Hybridization Washes 1-3

Tecan HS 400 Hybridization Station Hybridization

13.    Scan with Axon Genepix

14.    Load Gal file

15.    Find all the spots

16.    Check quality of spots

17.    Create output file

1.  Pour plates

NGM agar:

Mix the following:

3 g NaCl

17 g agar

2.5 g peptone

975 ml H20

Autoclave, and then sterilely add the following, mixing after each addition:

1 ml cholesterol (5 mg/ml in EtOH)

1 ml 1 M CaCl2

1 ml 1 M MgSO4

25 ml 1 M potassium phosphate pH 6

Pour ~ 3 liters to make ~120 100mmx15mm plates

Store at room temperature (bench top) for 4 days before spotting/spreading to allow the plates to dry.

2.  Spot/spread bacteria

1.    Using a sterile pipette, drop 1ml bacterial suspension on the middle of each 100 mm NGM plate.

2.    Take care not to damage the surface of the agar with the pipette tip (as worms will then burrow under the surface).

3.    Spread bacterial suspension uniformly over surface of the agar using a sterile glass spreader also taking care not to damage surface. 

4.    Let the plates sit 4 days to form a bacterial lawn before using.

3.  Clorox bleach worms and egg drop

See Decontamination with Clorox protocol

1.    From 4 plates with many eggs (~5 days after 3 L4Õs are placed on a plate) embryos are washed off with M9 and collected in a 15mL conical tube.

2.    Spin down tube and resuspend in a 20% Clorox, 0.5M NaOH (use M9 for dilution) solution for 6-10 minutes to release the eggs.  Halfway through treatment flick the tube to help dissipate worm bodies and release eggs.  Only treat past 6 minutes if bodies have not dissolved and do not treat past 10 minutes.

3.    Pellet for 4 minutes and resuspend in M9 then pellet again for 1 minute.

4.  Nematode Plate Wash

5.  RNA extraction

Yale RNA prep
(Rebecca D. Burdine, Michael J. Stern [modified by S.M. Hettenbach])

  1. Add 8 ml of TRIZOL to 2 ml packed worms in 15 ml centrifuge tube. Vortex and invert tube to solubilize and lyse worms for at least 10 min.
  2. Divide into two 15 ml c/f tubes--5 ml each.
  3. Invert for additional 5 min. Solution should be pink and will contain thread-like material. Spin at 4¡C, 4000 rpm for 20 min to remove insoluble material. All spins done in fixed angle rotor with white inserts.
  4. Transfer supernatant to new 15 ml c/f tubes(2). Add 800 l chloroform to each tube.
  5. Vortex 15 sec, incubate at room temperature for 3 min. Smeary preps will result if vortexing not long enough.
  6. Spin at 4¡C, 4000 rpm for 20 min to separate phases.
  7. Transfer upper phase to new 15 ml c/f tubes(2). Add 2 ml isopropanol to each tube.
  8. Invert to mix & incubate at RT for 10 min. Spin at 4¡C, 4000 rpm for 30 min.
  9. Remove s/n with pipet without disturbing the pellet.
  10. Add 400 ul 75% EtOH, vortex briefly.
  11. Spin at 4¡C, 4000 rpm for 10 min.
  12. Remove s/n with pipet and invert racked tubes in hood with door half shut. Approx. 10-15 minutes; do not overdry.
  13. Add 300 l DEPC-treated ddH2O to each tube and resuspend and combine tubes using wide opening tips. May have to heat briefly at 45-50¡C to completely dissolve. Really good yields may require adding more H2O.
  14. Use 2 l to do OD260 and OD280 to determine concentration. An A260/280 ratio of <1.6 indicates partially dissolved RNA. 1 OD260 is equal to 40 g/ml of RNA.
  15. Store at -80¡C in 1 mg aliquots, avoid repeat freezing and thawing.

6.  mRNA prep (optional)

Joe's mRNA prep
(Audrey Gasch, Pat Brown [modified by S.M. Hettenbach])

Oligo-dT cellulose prep

  1. Dump Ambion vial contents into 50 ml c/f tube
  2. Add 10 ml 1x NETS to vial, cap, rinse vial, dump 10 ml into c/f tube
  3. Spin tube 3000 rpm, 2 min at 4¡C in swinging bucket with no brake, remove s/n
  4. Add 10 ml 1x NETS to tube, resuspend contents by swirling
  5. Spin tube 3000 rpm, 2 min at 4¡C in swinging bucket with no brake, remove s/n
  6. Repeat steps 4 and 5 two more times for a total of 4 washes
  7. Add 10 ml 2x NETS and store at 4¡C
  8. Prep cellulose the day before you need it

mRNA prep

  1. Dilute 1 mg total RNA to 1 ml with 10mM Tris pH7.4 in c/f tube
  2. In a 2 ml BioRad mini column(#731-1550) sealed on bottom, mix 1 ml resuspended resin with 1 ml of 1 mg/ml total RNA from step 1. Cap and parafilm column, tape to rocker, and bind at RT for 1 hour on 5.5 setting
  3. Break bottom off column and place in 16 x125 mm test tube and allow contents to flow through
  4. Wash resin five times with 0.7 ml 1x NETS, slow flow is normal
  5. Transfer column to support and elute 2x with 0.7 ml 10 mM Tris pH 7.4 preheated to 70¡C into separate 1.7 ml tubes. Elute with 150 l of the 0.7 ml volume at a time keeping rest at 70¡C.
  6. Add 0.7 ml chloroform to each tube to remove residue cellulose, mix well, spin 13,000 rpm for 1 min at RT, transfer upper phase to new tube
  7. Precipitate with 0.1 volume 3M NaOAc pH 7.0 and 1 vol isopropanol at -20¡C for 1 hour
  8. Spin 13,000 rpm for 30 min at 4¡C
  9. Remove s/n and air dry
  10. Resuspend both tubes in final volume of 20 l of DEPC ddH2O
  11. Spec OD260 & OD280 on 1 l mRNA. 1 OD260 is equal to 40 g/ml of RNA
  12. Store at -80¡C, avoid repeat freezing and thawing

**Or just use QiagenÕs Oilgotex mRNA Midi Kit cat # 70042

7.  cDNA synthesis

For steps 6-11 See website: http://www.genisphere.com/pdf/array350_10_19_04.pdf

3DNA¨ Array 350ª

Expression Array Detection Kit for Microarrays

Cy3TM/Cy5TM Kit Cat No. W300180 5 Assays each

Preparation of cDNA from Total RNA (Reverse Transcription)

Since microarrays and RNA preparations vary in quality, the exact amount of RNA required for a given experiment will typically range from 1-5µg of animal total RNA or 2.5-10µg of plant total RNA. For new users, 5µg of animal total RNA or 10µg of plant total RNA is recommended as a starting point for cDNA synthesis. Larger or smaller amounts of RNA may be required to achieve optimal results, depending on the quality of the RNA sample and the array.

For use of higher quantities of RNA in large-scale cDNA synthesis, follow Appendix A.

For use of senseRNA or poly(A) RNA, follow Appendix B.

1. In a microtube, prepare the RNA-RT primer mix:

1-10µl total RNA (1-5µg mammalian total RNA or 2.5-10µg plant total RNA)

1µl RT primer (Vial 2, 1pmole/µl, either Cy3/Alexa Fluor 546 or Cy5/Alexa Fluor 647)

Add Nuclease Free Water (Vial 10) to a final volume of 11µl

2. Mix the RNA-RT primer mix and microfuge briefly to collect contents in the bottom of the tube.

3. Heat to 80¡C for 10 minutes and immediately transfer to ice for 2-3 minutes.

4. In a separate microtube on ice, prepare a reaction mix for every RT reaction:

4 µl 5X SuperScript II First Strand Buffer (or equivalent reaction buffer supplied with enzyme)

2 µl 0.1M dithiothreitol (if supplied with enzyme; otherwise use Nuclease Free Water (Vial 10))

1 µl dNTP mix (Vial 3)

1 µl Superase-Inª RNase inhibitor (Vial 4)

1 µl Superscript II enzyme, 200 units (or equivalent reverse transcriptase)

5. Gently mix (do not vortex) the reaction mix and microfuge briefly to collect reaction mix contents in the bottom of

the tube. Keep on ice until used.

6. Add the 9µl of reaction mix from step 5 to the 11µl of RNA-RT primer mix from step 3 (20µl volume).

7. Gently mix (do not vortex) and incubate at 42¡C for 2 hours.

8. Stop the reaction by adding 3.5µl of 0.5M NaOH/50mM EDTA.

9. Incubate at 65¡C for 15 minutes to denature the DNA/RNA hybrids and degrade the RNA.

10. Neutralize the reaction with 5µl of 1M Tris-HCl, pH 7.5.

11. For Single Channel Assays:

Add 101.5µl of 1X TE buffer to the reaction from step 10 (130µl volume).

For Dual Channel Assays:  Combine the Cy3/Alexa Fluor 546 and Cy5/Alexa Fluor 647 reactions from step 10 into one tube.

Rinse the empty tube with 73µl of 1X TE buffer.

Combine the rinse with the reaction mixture (130µl volume).

The steps 8-12 are for manual hybridization

8.  Prehybridization of array

Array Prewashing Procedure

Prewashing the array can help to remove salts and other debris that can become immobilized during hybridization,

resulting in high background.

1. Wash the microarray by the following conditions:

a. 2X SSC/0.2% SDS for 20 minutes at 55¡C

b. 0.2X SSC for 5 minutes at room temperature

c. Deionized distilled water for 3 minutes at room temperature

2. Immediately transfer the array to a dry 50 mL centrifuge tube. Do this quickly to avoid streaky background on the slide. Orient the slide so that any label is at the bottom of the tube. Centrifuge without the tube cap for 2 minutes at 800-1000 RPM to dry the slide. Avoid contact with the array surface. The array is now ready for either prehybridization or hybridization with cDNA.

Array Prehybridization Procedure

Prehybridization of the array with BSA can help reduce some types of nonspecific binding, a common cause of high

background. 2X Formamide-Based Hybridization Buffer (Vial 7) contains BSA and can be used to pre-coat the array.

1. Prewarm the microarray to 50¡C for 10 minutes.

2. Thaw and resuspend the 2X Formamide-Based Hybridization Buffer (Vial 7) by heating to 70¡C

for at least 10 minutes or until completely resuspended. Vortex to ensure that the components are resuspended evenly. If necessary, repeat heating and vortexing until all the material has been resuspended. Microfuge for 1 minute.

3. Prepare the Prehybridization Mix as follows:

25µl 2X Formamide-Based Hybridization Buffer (Vial 7)

1µl Human Cot-1 DNA

24µl Nuclease free water

4. Heat the Prehybridization Mix to 80¡C for 10 minutes.

5. Apply the Prehybridization Mix to the prewarmed microarray and cover with a 24x60mm coverslip.

6. Incubate at 50¡C for 1-2 hours.

7. Wash the array by the following conditions:

a. 2X SSC, 0.2% SDS for 15 min at 60-65¡C.

b. 2X SSC for 10 min at room temperature.

c. 0.2X SSC for 10 min at room temperature.

8. Immediately transfer the array to a dry 50 mL centrifuge tube. Do this quickly to avoid streaky background on the slide. Orient the slide so that any label is at the bottom of the tube. Centrifuge without the tube cap for 2 minutes at 800-1000 RPM to dry the slide. Avoid contact with the array surface. The array is now ready for hybridization with cDNA.

9.  Hybridization of cDNA

cDNA Hybridization and Wash

cDNA Hybridization

1. Thaw and resuspend the hybridization buffer (see Hybridization Conditions, pg. 7, for help in selecting the appropriate buffer) by heating to 65-70¡C for at least 10 minutes or until completely resuspended. Vortex to ensurethat the buffer is resuspended evenly. If necessary, repeat heating and vortexing until all the material has been resuspended. Microfuge for 1 minute.

2. For each array, prepare a cDNA Hybridization Mix according to the tables below: Optional: 1.0µl Cot-1 DNA may be added to the cDNA Hybridization Mix. Cot-1 DNA (species specific) should be denatured at 95-100¡C for 10 minutes prior to use. Note: Enhanced Hybridization Buffer (Vial 12) requires higher hybridization volumes due to its increased viscosity.

Option 1 (Recommended): Use of Enhanced Hybridization Buffer (Vial 12)

Option 2: Use of Vial 6 or Vial 7 Hybridization Buffers

3. Gently vortex and briefly microfuge the cDNA Hybridization Mix. Incubate the cDNA Hybridization Mix first at 75-80¡C for 10 minutes, and then at the hybridization temperature until loading the array (see the table located below step 5 for recommended hybridization temperatures). Pre-warm the microarrays to the hybridization temperature.

4. Gently vortex and briefly microfuge the cDNA Hybridization Mix. Add the cDNA Hybridization Mix to a prewarmed microarray, taking care to leave behind any precipitate at the bottom of the tube.

5. Apply a glass coverslip to the array. Incubate the array overnight in a dark humidified chamber at the appropriate hybridization temperature:

Spotted DNA Size        Vial 6 or 12 Buffer       Vial 7 Buffer

30mer                           42-47¡C                      30-35¡C

50mer                           55-60¡C                      43-48¡C

70 mer                         55-62¡C                      43-50¡C

PCR Product (cDNA)      55-65¡C                      43-53¡C

The hybridization temperatures recommended in this protocol are intended as a starting point and should be used as a guide for further optimizations. It may be necessary to adjust the temperatures to meet the stringency requirements dictated by the nature of the nucleic acids spotted on the array as well as the slide surface chemistry. In particular, increasing the hybridization temperature by 5¡C may remove non-specific signal.

10.  Post cDNA Hybridization Washes 1-3

Post cDNA Hybridization Wash

1. Prewarm the 2X SSC, 0.2%SDS wash buffer to:

¥ 65¡C for PCR product (cDNA) arrays

¥ 42¡C for oligonucleotide spotted arrays

2. Remove the coverslip by washing the array in prewarmed 2X SSC, 0.2% SDS for 2-5 minutes or until the coverslip floats off.* Additional time may be required to remove the coverslip when the 2X Enhanced cDNA Hybridization Buffer (Vial 12) is used.

3. Wash for 15 minutes in prewarmed 2X SSC, 0.2%SDS.

4. Wash for 10-15 minutes in 2X SSC at room temperature.

5. Wash for 10-15 minutes in 0.2X SSC at room temperature.

6. Transfer the array to a dry 50mL centrifuge tube, orienting the slide so that any label is at the bottom of the tube. Immediately centrifuge without the tube cap for 2 minutes at 800-1000 RPM to dry the slide (any delay in this step may result in high background). Avoid contact with the array surface.  Further optimization of wash conditions may be required to achieve optimal array performance. If necessary to reduce background on the array, we recommend increasing the time of some or all of the washes to 15-20 minutes. Agitation during washing may also help to reduce background due to non-specific binding to the surface of the array.

Proceed to Step 4: 3DNA Hybridization and Wash, pg. 14.

*Note: If the coverslip is difficult to remove, this may be an indication of drying. To prevent this problem from recurring in future experiments, increase the total volume of the cDNA

Hybridization Mix by adding equal volumes of Nuclease Free Water (Vial 10) and 2X Hybridization Buffer (Vial 6, 7 or 12). In addition, ensure that the hybridization chamber is

properly humidified and sealed.

11.  Hybridization of 3DNA

Step 4: 3DNA Hybridization and Wash

3DNA Hybridization

1. Prepare the 3DNA Array 350 Capture Reagent (Vial 1). It is necessary to break up aggregates that may form as a result of the freezing process.

a. Thaw the 3DNA Array 350 Capture Reagent (Vial 1) in the dark at room temperature for 20 minutes.

b. Vortex at the maximum setting for 3 seconds and microfuge briefly.

c. Incubate at 50-55¡C for 10 minutes.

d. Vortex at the maximum setting for 3-5 seconds.

e. Microfuge the tube briefly to collect the contents at the bottom.

Be sure to check the sample for aggregates prior to use and repeat vortex mixing if necessary. Aggregates may appear as small air bubbles or flakes at the side of the tube below the surface of the solution. Repeat steps a-e if necessary.

2. Thaw and resuspend the 2X Hybridization Buffer (Vial 6 or Vial 7) by heating to 70¡C for at least 10 minutes or until completely resuspended. Vortex to ensure that the components are resuspended evenly. If necessary, repeat heating and vortexing until all the material has been resuspended. Microfuge for 1 minute. Warning: Do not use the 2X Enhanced cDNA Hybridization Buffer (Vial 12) in the 3DNA Hybridization step.

3. Optional: Prepare a stock solution of Anti-Fade Reagent (Vial 8) by combining 1µl of Anti-Fade with 100µl of either of the 2X Hybridization Buffers to be used in the 3DNA hybridization (Vial 6 or Vial 7). The Anti-Fade Reagent helps reduce fading of the fluorescent dyes both during and after the hybridization process. Store any unused hybridization buffer containing Anti-Fade Reagent at –20oC and use within two weeks of preparation. Do not use Anti-Fade Reagent on arrays printed on aldehyde-coated glass, as this may cause high background.

4. For each array, prepare a 3DNA Hybridization Mix according to the table below. Optional: 1.0µl Cot-1 DNA may be added to the cDNA Hybridization Mix. Cot-1 DNA (species specific) should be denatured at 95-100¡C for 10 minutes prior to use. Note: For single channel expression analysis, use 2.5µl of Nuclease Free Water (Vial 10) in place of the second 3DNA Capture Reagent.

5. Gently vortex and briefly microfuge the 3DNA Hybridization Mix. Incubate the 3DNA Hybridization Mix first at 75-80¡C for 10 minutes, and then at the hybridization temperature until loading the array (see the table located below step 7 for recommended hybridization temperatures). Pre-warm the microarrays to the hybridization temperature.

6. Gently vortex and briefly microfuge the 3DNA Hybridization Mix. Add the 3DNA Hybridization Mix to a prewarmed

microarray, taking care to leave behind any precipitate at the bottom of the tube.

7. Apply a glass coverslip to the array. Incubate the array for 3-4 hours in a dark humidified chamber at the appropriate hybridization temperature:

Spotted DNA                Vial 6 Buffer                Vial 7 Buffer

Oligonucleotide              55-65¡C                       43-53¡C

PCR Product (cDNA)       60-65¡C                       48-53¡C

12.   Post 3DNA Hybridization Washes 1-3

Post 3DNA Hybridization Wash

Perform the following steps in the dark to avoid degradation and fading of the fluorescent dyes. Please refer to Appendix E for recommendations for reducing the degradation of Cy5 / Alexa Fluor 647.  (Not as touchy as is implied but try not to be in direct light, however some light is necessary to avoid pipetting errors etc.)

1. Prewarm the 2X SSC, 0.2%SDS wash buffer as follows:

¥ 65¡C for PCR product (cDNA) arrays and oligonucleotide arrays greater than 50 nucleotides long

¥ 42¡C for oligonucleotide arrays less than 50 nucleotides long

2. Remove the coverslip by washing the array in prewarmed 2X SSC, 0.2% SDS for 2-5 minutes or until the coverslip floats off.*

3. Wash for 15 minutes in prewarmed 2X SSC, 0.2%SDS.

4. Wash for 10-15 minutes in 2X SSC at room temperature.

5. Wash for 10-15 minutes in 0.2X SSC at room temperature.

6. Transfer the array to a dry 50mL centrifuge tube, orienting the slide so that any label is at the bottom of the tube.  Immediately centrifuge without the tube cap for 2 minutes at 800-1000 RPM to dry the slide (any delay in this step may result in high background). Avoid contact with the array surface.  Further optimization of wash conditions may be required to achieve optimal array performance. If necessary to reduce background on the array, increase the time of some or all of the washes to 15-20 minutes. Agitation during washing may also help to reduce background due to non-specific binding to the surface of the array.

Proceed to Signal Detection, or first apply DyeSaver 2 coating (Genisphere Cat No. Q500500) to preserve fluorescent signal and reduce photo-bleaching.

*Note: If the coverslip is difficult to remove, this may be an indication of drying. To prevent this problem from recurring in future experiments, increase the total volume of the 3DNA Hybridization Mix by adding equal volumes of Nuclease Free Water (Vial 10) and 2X Hybridization Buffer (Vial 6 or 7). In addition, ensure that the hybridization chamber is properly humidified and sealed.