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Core Microarray Standard Operating Procedure
RNA Quality Control:
1. In a 50 ul PCR reaction, test RNA for genomic DNA contamination, using 100300 ng RNA.
a. Mix per rxn:
5 ul 10X PCR buffer
2.5 ul primer 1 (10 pmol stock)
2.5 ul primer 2 (10 pmol stock)
1.25 ul 10 mM dNTP
0.25 ul Taq polymerase (5 U/ ul)
Nuclease-free H2O to 50 ul
b. Program (BRAD2):
i. 95oC 3 min
ii. 94oC 15 sec
iii. 55-58oC* 30 sec
iv. 72oC 60-90 sec*
v. 40 cycles steps 2-4
vi. 72oC 10 minutes
vii. 4oC forever
* annealing temperature and extension times will vary depending
on primer Tm and size of PCR product (1 minute/1 kb of product)
c. Primers:
i. For G. sulfurreducens QC, use primers hfqC and hfqD, hfqA and
hfqB, or 1734F and 1734R (last set gives small, ~100 bp, band, so
the others are preferred).
ii. For other organisms, use the ones provided by the researcher
submitting the RNA.
d. Run a negative and positive (genomic DNA) PCR control.
2. If DNA is still present, as represented by a PCR band, treat sample with DNAfree.
a. Set up reaction based on volume of RNA sample:
i. Add 10X DNA-free Buffer to make a 1X concentration, adjusting
with nuclease-free water if necessary.
ii. For a 50-100 ul volume, add 2 ul DNA-free. For a 500 ul volume,
add 5 ul DNA-free.
iii. Mix well.
iv. Incubate at 37 C 30 minutes, ice 1 minute.
v. Add 20 ul inactivation reagent
vi. Flick to mix
vii. Incubate 5 min at room temperature, flicking again ½ way through
incubation.
viii. Spin 5 minutes
ix. Move RNA containing supernatant to as fresh labeled tube.
3. Repeat Step 1 and 2 until RNA is clean (no PCR product from the RNA sample)
4. On a 1.2 % agarose gel, run out 500 ng RNA.
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a. Ribosomal bands should look sharp
b. The 23S band should not look fainter than the 16S band.
c. A stronger 23S band is a sign of RNA degradation.
If the RNA is below 50 ug, consider RNA amplification and see SOP
RNA Labeling:
1. In a PCR tube combine:
a. 10 ug RNA
b. 2-6 ul ASAP Labeling Reagent (typically 6 ul) Use the same amount for
each sample in an experiment set
c. 2 ul Cy3 (control) or Cy5 (experiment) reagent
d. Nuclease-free H2O to 20 ul.
2. Mix by pipetting up and down.
3. Incubate in thermocycler for 15 minutes at 85oC.
4. Add 180 ul nuclease-free H2O
5. Place in Microcon Y-30 filter column assembled in a collection tube.
6. Spin at 25oC for 8.5 minutes at full speed.
7. Add 200 ul H2O to Microcon Y-30 filter column
8. Pipet up and down to resuspend labeled RNA
9. Spin at 25oC for 8.5 minutes at full speed.
10. Repeat steps 7-9.
11. Add 8 ul nuclease-free H2O to each Microcon Y-30 filter column to resuspend the
labeled RNA
12. Place each Microcon Y-30 filter column upside down in a new collection tube.
13. Spin at 25oC for 2 minutes at 10,000xg (~ 10,600 rpm)
14. Remove the Microcon Y-30 filter column from the tube
15. Tlace the tube on ice
16. Check the RNA using Microarray setting on the Nanodrop
a. good label incorporation is generally between 18-35 pmol/ul, but you can
work with slightly lower incorporation
RNA Fragmentation and Precipitation:
1. Transfer RNA to PCR tubes
2. Add nuclease-free H2O to bring up the volume of the labeled RNA to 18 ul.
3. Add 2 ul fragmentation reagent to each tube.
4. Incubate in a thermocycler at 70oC for 30 minutes.
5. Combine Cy3 and Cy5 labeled RNA for each hybridization in a 1.5 ml screw-cap
tube (~40 ul volume)
6. Add 5 ul 5M NH4OAc and 135 ul chilled 95 % EtOH (stored at -20 C).
7. Vortex to mix, incubate 1 hour at -20oC.
8. Spin at 4oC for 30 minutes at full speed.
9. Remove supernatant with pipettor
10. Wash 3X with 150 ul 70 % EtOH (made from 95% stock – do not use 100%
stock).
11. Let air-dry 5-10 minutes, being sure to cover tubes with blue pipet tip box cover
to prevent photobleaching.
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12. Resuspend labeled RNA with 18 ul H2O.
13. Add 80 ul Hybridization Solution to each tube
14. Resuspend by pipetting up and down.
15. Proceed to Hybridization Protocol, or alternatively store probe at -20oC. RNA is
stable for several weeks at -20oC.
Hybridization Protocol:
1. Prehybridizing arrays can be started while labeled RNA is precipating.
a. Make Hybridization Solution (Per hybridization):
i. 50 ul 2X Hybridization Solution
ii. 25 ul DI Formamide (Ambion)
iii. 1 ul salmon sperm DNA (Sigma)
iv. 4 ul 1% SDS
v. Vortex to mix.
b. Make Prehybridization Solution (Per slide):
i. 50 ul 2X Hybridization Solution
ii. 10 ul 50X Denhardt’s Solution (Sigma)
iii. 1 ul salmon sperm DNA (Sigma)
iv. 5 ul 1% SDS
2. Set hybridization oven at 65oC.
3. Assemble Combimatrix slides with hybridization chambers (note: no disassembly
will be required until you are ready to scan).
4. Add 100 ul nuclease-free H2O to each slide through one of the two ports on
hybridization chamber.
5. Rotate the slide at 65oC in the hybridization oven for 10 minutes (This step
removes oxygen from slide, which could otherwise inhibit hybridization)
6. Set oven temperature to 45oC, and leave door open to let temperature decrease.
7. Remove H2O from slide through the two ports
8. Pipet 100 ul prehybridization solution into chamber through one port, leaving a
small air bubble.
9. Seal gaskets with adhesive seal tabs (Invitrogen).
10. Let slides rotate for at least 30 minutes at 45oC.
11. After at least 30 minutes of prehybridization, place labeled RNA (in hybridization
buffer) in a PCR tube
12. Heat at 95oC for 3 minutes in a thermocycler.
13. Ice RNA for 2 minutes.
14. Remove slides from oven and remove adhesive tabs from ports.
15. Replace prehybridization solution with hybridization mix (containing labeled
RNA).
16. Add adhesive tabs
17. Let slides rotate overnight at 45oC.
18. Place Wash1 (50 ml tube) in hybridization oven overnight sealed with Parafilm or
for at least 30 minutes (needs to be at 45oC for first wash).
Note: Make fresh wash solutions if more than two weeks old (Combimatrix
protocol).
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Slide Washing:
Turn on GenPix 4000B scanner to allow the instrument to warm up during the wash
protocol
1. Remove slides from oven
2. Remove the probe and save in a new tube.
3. Add 100 ul 45oC Wash 1 solution
4. Remove Wash 1 solution
5. Add another 100 ul 45oC Wash 1 solution
6. Seal ports and rotate in oven at 45oC for 5 minutes.
7. Remove Wash 1 solution
8. Add 100ul Wash 2 solution, remove
9. Remove Wash 2 solution
10. Add another 100 ul Wash 2 solution
11. Lay slides flat (chamber side up) in a drawer for 5 minutes.
12. Remove Wash 2 solution
13. Repeat Steps 8-12 with Wash solutions 3-5
14. Repeat Steps 8-11 with Wash solution 5 a second time.
15. Remove Wash solution 5
16. Add 100 ul Visualization Solution
17. Disassemble the first slide, while leaving the other slides in the drawer.
18. Place a LifterSlip on top of the array, avoiding air bubbles (Use either a Lifterslip
that comes shipped with arrays or a 22x30 Lifterslip – I prefer the 22x30 slips).
Slide Scanning:
Before using software for scanning, see Ray for proper training
1. Open GenePix software
2. Adjust settings:
a. Use 5 pixels
b. 532 nm scan setting at 450
c. 635 nm scan setting at 450.
d. Select settings file for array.
3. Place slide Lifterslip facing down into scanner.
4. “Pre-scan” slide by clicking the double-arrow icon.
5. Select scan area
6. “Scan” slide by clicking the single arrow icon
7. Adjust scan settings for 532 or 635 until overall slide image has no appreciable
green or red background (this is more to assist the eye, the software and statistical
analysis subtract any background)
8. If adjustments were made, scan image again such that entire image was scanned at
one setting.
9. Save Image (Multi-Image TIFF) as “Slide Number YYYY-MM-DD”.
10. Focus in on image
11. Select “Settings” icon
12. Move grid onto array image
13. Line up grid with spots. Begin at each corner, while holding control, drag until
grid lines up with spots. Repeat for all four corners of image.
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14. Mark any “bad spots”, lint or other oddities.
15. Save settings file (.GPS), as “Slide Number MMDDYY”.
16. Hit “Analyze” icon.
17. Save Results File (.GPR) as “Slide Number YYYY-MM-DD”
Analysis in Acuity4.0:
Acuity4.0 is sofware which allows for easy visualization of array data and has a
normalization feature which allows for preliminary data analysis for experimental
consistency and quality analysis.
If something is unclear in this protocol, see Ray or go to Acuity Help
1. Open Acuity, User “sa”, Password, “shadow”.
2. Under “File”, go to “New Folder”. Name it for your experiment and deposit it in
appropriate already existing folder.
3. Under “File”, go to “Import Microarrays”.
4. Select GPR files under Axon, Data, Your files”. You can select multiple files
using the Ctrl key.
5. After importing arrays, go to Analysis, Normalization, Normalization Wizard.
6. Follow instructions, selecting GPR files from the folder they were imported into,
and hit OK.
7. Select Lowess Normalization
8. Select Print-tip to be “off”
9. Hit Analyze.
10. After viewing results of normalization, close.
11. Select your GPR files in their Acuity folder, select “Open”, under “File”.
12. Array data will open
13. Select Lowess M Normalization under pull down menu
14. Select all array data columns
15. Go to statistics, hit F5.
16. Click on Mean to sort by mean
17. Go back to data.
18. To view annotations, select annotation.
19. Go to Substance Annotations to Retrieve, select your annotation and which fields
you would like to view, hit OK.
20. Annotation fields should be updated, or you may have to press “Refresh
Columns” icon.
Stripping Slides:
Usually, an experiment can be run using three Combimatrix 12K slides, stripping the
slides before hybridizing the second technical replicate.
1. Preheat hybridization oven to 70oC.
2. Rinse slides in a storage container with nuclease-free water.
3. Reassemble hybridization chambers onto slides
4. Rinse 3 times with nuclease-free water
5. Add 100 ul nuclease-free water
6. Rotate for 10 minutes at 70oC.
7. Add Stripping Solution
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8. Mix well by pipetting, while avoiding numerous air bubbles
9. Incubate at 70oC 1 hour.
10. Place slides in storage container with nuclease-free water
11. Rinse 3 times
12. Add imaging solution and LifterSlip
13. Scan at scan settings of 500 for 532 and 635 nm.
14. If there are noticeable spots, repeat steps 2-13.
15. Store slides in 2X PBS at 4oC for up to two weeks, or reassemble hybridization
chamber and proceed with Hybridization Protocol.