ORNL MICROARRAY HYBRIDIZATION PROTOCOLS

Direct labeling of total RNA with Cy3 and Cy5:

A. MATERIALS

RNeasy® Mini Kit (Qiagen; Cat # 74106)
SuperScript II RT (200U/µL) (Life Technologies; Cat # 18064-014)
QIAquick PCR Purification Kit (Qiagen; Cat # 28106)
100 mM dNTP Set PCR grade (Life Technologies; Cat # 10297-018)
Primers:   Poly d(T) [20-mers] 2.5 mg/mL (avoids labeling the gDNA and rRNA.)
                Anchored poly d(T) primers
           (Archaic) Random Hexamer primers (3mg/mL) (Life Technologies; Cat # 48190-011)
Cyanine 3-dUTP # NEL578  or Cyanine 5-dUTP #NEL579 (Perkin Elmer/NEN)

50X dNTPs

	50X [ ]	Stock [ ]	For 1.0 ml of 50X use:
dTTP	5 mM	100mM	50 µL
dATP	25mM	100mM	250 µL
dGTP	25mM	100mM	250 µL
dCTP	25mM	100mM	250 µL
RF-ddH20			200 µL

 

Master Mix:	(Per Reaction)	USED TODAY:
5X Enzyme Buffer	6 µL
0.1 M DTT	3 µL
50X dNTPs	0.6 µL
Superscript II RT	2 µL

C. PROCEDURE: This protocol uses 30-100µg of total RNA (Prepare two 30-100 µg samples for comparative hybridizations using two-colors). To use the volumes listed, the RNA must be at ? 2.5 mg/ml. Protect slide from light at all times until imaged to prevent bleaching of dyes.

1. Annealing Primer to RNA:

1.1. Dispense 30-100µg of total RNA (12.9 µL total) into screw-cap tube
1.2. Add 2.5 µL of oligo dT (2.5 mg/ml)
1.3. Adjust volume to 15.4 µL with DEPC water.
1.4. Heat to 70°C for 10 min to denature
1.5. immediately transfer to ice for > 2 min.

2.1. To each sample add 3.0 µL of either Cy-3 (pink)  or Cy-5 (blue) dUTP (NEN).
2.2. To each sample add 11.6 µL of master mix (see above).
2.3. Incubate 1 hr at 42°C.
2.4. Add 1 µL of SuperScript II Reverse Transcriptase (GIBCO/Life Technologies) to each tube and incubate another 1 hour at 42°C.
2.5. Remove samples to ice.
2.6. Add 1.5 µL of 20mM EDTA to stop reaction
2.7. Add 7.5 µL of 0.1 M NaOH. Heat to 70°C for 10 min to hydrolyze the RNA.
2.8. Cool to RT° and add 7.5 µL of 0.1 M HCl to neutralize the sample. (The HCl step can be skipped if using QIAQUIK columns below)
2.9. Volume now should be 47 µL per sample

3.1. Add 237.5 µL (5 volumes) of Buffer PB to samples.
3.2. Place onto QIAquick column.
3.3. Spin 30-sec @ 12K. Discard filtrate.
3.4. To column, add 700 µL PE. Spin 30 sec @ 12K. Discard filtrate
3.5. To column, add 700 µL PE. Spin 30 sec @ 12K. Discard filtrate.
3.6. Re-spin column to dry column bed.
3.7. Move column to fresh µ-fuge tube
3.8. Add 30 µL EB to center of column bed. Spin 30 sec @ 12K. Do not change tube.
3.9. Add another 30 µL EB to center of column bed. Spin 30 sec @ 12K. Save pooled eluant.
3.10. Mix the differently labeled samples (e.g. Cy-3 and Cy-5) at this point.
3.11. Remove 1-2 µL of sample to check RT-reaction on agarose gel (Optional)

4.1. To each mixed sample, add .2 µL of polydA (1mg/ml), and 1 µL of Cot-1 DNA (1mg/ml)
4.2. Dry samples in Speedvac using medium setting. Do NOT use “High” setting which turns on heat light. Should take about 15-30min. Dried samples can be temporarily stored, wrapped in foil, frozen at -80°C.
4.3. Begin Prehybridization of arrays while samples are drying
4.4. Dried samples should be resuspended in hybridization buffer* then used immediately.
4.5  Continue with the ORNL AT Hybridization Protocol to apply the probe to a microarray.

Secure the top of the microfuge tube with a clip, and heat to 95°C for 5 min to denature.
Centrifuge 2 minutes. Do not transfer to ice.

Apply targets to the microarray, avoiding bubbles. Place coverslip slowly down onto array. If bubbles form, do not attempt to "push" them out. This will damage your array.
Hybridize the array in a humidified container overnight (16-18 hours) in a 42°C water bath.
 (floating is usually OK, but can be placed on a support to keep it level)

After hybridization, remove slide from Hybridization chamber (avoid water dripping onto array).

Wash array:

1 X 5min in 2X SSC, 0.1% SDS @ 42°C (37-42°C is OK)
2 X 5min in 0.2X SSC at RT°
4 X 1min in 0.1X SSC at RT°

Protect slide from light at all times until imaged to prevent bleaching of dyes. 
  
 

Indirect labeling of RNA using aminoallyl-modified nucletides and monoester reactive Cy-dyes

1. MATERIALS

1.1 5-(3-aminoallyl)-2’deoxyuridine-5’-triphosphate (AA-dUTP) (Sigma; Cat # A0410)
1.2 100 mM dNTP Set PCR grade (Life Technologies; Cat # 10297-018)
1.3 Primers
1.3.1 Poly d(T) [20-mers] 2.5 mg/mL used instead of random primers to avoid labeling the gDNA and rRNA.
1.3.2  Anchored poly d(T) primers
1.3.3  (Archaic) Random Hexamer primers (3mg/mL) (Life Technologies; Cat # 48190-011)
1.4 SuperScript II RT (200U/µL) (Life Technologies; Cat # 18064-014)
1.5 Cy-3 ester (AmershamPharmacia; Cat # PA23001: 100,000 picomoles dye/tube)
1.6 Cy-5 ester (AmershamPharmacia; Cat # PA25001: 100,000 picomoles dye/tube)
1.7 Monoreactive dye pack for aminoallyl labeling (Amersham Cat # RPN5661: 40,000 picomoles dye/tube. Cy3 and Cy5)
1.7 QIAquick PCR Purification Kit (Qiagen; Cat # 28106)
1.8 RNeasy® Mini Kit (Qiagen; Cat # 74106)
1.9 Microcon YM-30 Centrifugal Filter Devices (Millipore; Cat # 42410)
1.10 Anhydrous DMSO (EM Scientific, "DriSolv™",  #EM-MX1457-6 from VWR)

2. REAGENT PREPARATION

2.1 Phosphate Buffers

2.1.1 Prepare 2 solutions: 1M K2HPO4 and 1M KH2PO4
2.1.2 To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine:

1M K2HPO4   9.5 mL
1M KH2PO4  0.5 mL
(Store 1M KPO4 (pH 8.5) @-20°C in 10ml aliquots)

2.1.3 For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix:

1 M KPO4 pH 8.5 0.5 mL
MilliQ water  15.25 mL
95% ethanol  84.25 mL
Note: Wash buffer will be slightly cloudy at first. Store Phosphate wash buffer @ room temperature.

2.1.4  Phosphate elution buffer (PEB) is made by diluting 1 M KPO4, pH 8.5 to 4 mM with MilliQ water. Make 10 ml, aliquot into 1.0 ml, and store @-20°C
2.1.5 To prepare  0.1 M KPO4 (pH=7.5) mix, then autoclave:

 1M K2HPO4  41.5 mL
 1M KH2PO4  8.5 mL
 MilliQ water  450 mL

2.2 Aminoallyl dUTP

2.2.1 For a final concentration of 100 mM add 19.1 µL of 0.1 M KPO4 buffer (pH 7.5) to a stock vial containing 1 mg of aa-dUTP. Gently vortex to mix and transfer the aa-dUTP solution into a new microfuge tube. Store at –20°C.
2.2.2 Measure the concentration of the aa-dUTP solution by diluting an aliquot 1:5000 in 0.1 M KPO4 (pH 7.5) and measuring the OD289. (Stock concentration in mM = OD289 x 704)

2.3 Labeling Mix (50X) with 2:3 aa-dUTP: dTTP ratio

2.3.1 Mix the following reagents:

 

Stocks	Mix:	Final Concentration
dATP (100 mM)	5 µL	(25 mM)
dCTP (100 mM)	5 µL	(25 mM)
dGTP (100 mM)	5 µL	(25 mM)
dTTP (100 mM)	3µL	(15 mM)
aa-dUTP (100 mM)	2µL	(10 mM)
Total:	20µL
Note: Varying the ratio of aa-dUTP to dTTP will affect incorporation

2.3.2 Store unused solution ("aa-dNTP Mix") at –20°C.

2.4 Sodium Carbonate Buffer (Na2CO3): 1M, pH 9.0

2.4.1 Dissolve 10.8 g Na2CO3 in 80 mL of MilliQ water
2.4.2 Adjust pH to 9.0 with 12 N HCl, then bring volume up to 100 mL with MilliQ water.
2.4.3 Aliquot into 1.0 mL tubes and freeze at -80°C. Use each tube for only 1-2 weeks.
Note: Carbonate buffer changes composition over time; aliquots stored at -80°C sould be made fresh every couple of months.

2.5 Cy-dye esters

2.5.1 Cy3-ester and Cy5-ester are provided From Amersham (# RPN5661) as a dried product in 12 tubes each.
2.5.2  Resuspend a tube of dye ester in 7.5 µL of anhydrous DMSO.
2.5.3  Aliquot 2.5 mL (approximately 13,000 picomoles) into 3 screw-cap tubes and place at -80°C in a light-proof rack with desiccant. This will allow you to use each packet three times.

2.5.3.1  For the PA23001 and PA25001 dye packets, resuspend in 20 µL and aliquot 2.5 µL (12,500 picomoles) into 8 tubes.

2.5.4 Wrap all reaction tubes with foil and keep covered as much as possible in order to prevent photobleaching of the dyes.
Note: Any water introduced to the dye esters will result in a lower coupling efficiency due to the hydrolysis of the dye esters. Because anhydrous DMSO is hygroscopic (absorbs water from the atmosphere), store DMSO well sealed at -80°C in desiccant unless a dry atmosphere can be maintained in the stock bottle.

3. PROCEDURE

3.1 Aminoallyl Labeling

3.1.1 To 10-20 µg of high-quality (e. g. CsCl-isolated, DNAse-treated, or QIAGEN RNAeasy purified) total RNA (or 2 µg poly(A+) RNA), add 2.5 µL oligo d(T) or "anchored-T" primers (2.5mg/mL) and bring the final volume up to 18.5 µL with RNase-free water.
3.1.2 Mix well and incubate at 70°C for 10 minutes.
3.1.3 Immediately place in an ice-water bath for 60 seconds, then centrifuge briefly at >10,000 rpm @ 4°C.
3.1.4 Thaw the following on ice and then add to each tube:

 

5X First Strand buffer	6 µL
0.1 M DTT	3 µL (Vortex after thawing)
50X aminoallyl-dNTP mix	0.6 µL
SuperScript II RT (200U/µL)	2 µL (Keep @ -20°C until use)

3.1.5 Mix and incubate at 42°C for 3 hours to overnight.
3.1.6 To hydrolyze RNA, thaw the following and add:

0.5 M EDTA 10 µL
1 M NaOH 10 µL

3.1.7 mix and incubate at 65°C for 15 minutes or 70°C for 10 minutes.
3.1.8 Add 10 µL of 1 M HCl to neutralize pH. (Alternatively, one can add 25 µL of 1 M HEPES pH 7.0 or 25 µL of 1 M Tris pH 7.4)

3.2 Reaction Purification I: Removal of unincorporated aa-dUTP and free amines (use either the Qiagen or the Microcon method, not both)

Note: This purification protocol is modified from the Qiagen QIAquick PCR purification kit protocol. The phosphate wash and elution buffers (prepared in 2.1.3 & 2.1.4) are substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines which compete with the Cy dye coupling reaction.

3.2.1 Mix cDNA reaction with 300 µL (5X reaction volume) buffer PB (Qiagen supplied) and transfer to QIAquick column.
3.2.2 Place the column in a 2 ml collection tube (Qiagen supplied) and centrifuge at ~13,000 rpm for 1 minute. Empty collection tube.
3.2.3 To wash, add 700 µL phosphate wash buffer to the column and centrifuge at ~13,000 rpm for 1 minute.
3.2.4 Empty the collection tube and repeat the wash and centrifugation step (5.2.3).
3.2.5 Empty the collection tube and centrifuge empty column an additional 1 minute at maximum speed.
3.2.6 Transfer column to a new 1.5 mL microfuge tube and carefully add 30 µL phosphate elution buffer (see 4.1.4) to the center of the column membrane.
3.2.7 Incubate for 1 minute at room temperature.
3.2.8 Elute by centrifugation at ~13,000 rpm for 1 minute.
3.2.9 Elute a second time into the same tube by repeating steps 5.2.6- 5.2.8. The final elution volume should be ~60 µL.
3.2.10 Dry sample in a speed vac protected from light. This is a STOPPING POINT

3.2.1 Place the Microcon sample reservoir into a collection microfuge tube. Add 375 µL of water to the cDNA reaction tube and then pipette the sample into the Microcon sample reservoir/collection microfuge tube without touching the membrane.
3.2.2 Centrifuge at 12,000 rpm for 6-10 min.
Note: Never centrifuge column to dryness as this will decrease product recovery. Adjust spin time to allow for optimal filtration while allowing enough solution to remain for sufficient recovery.
3.2.3 Empty collection microfuge tube.
3.2.4 To wash add 450 µL of water to the sample reservoir/collection microfuge tube and centrifuge at 12,000 rpm for 6-10 minutes.
3.2.5 Empty collection microfuge tube and repeat previous wash step.
3.2.6 Invert Microcon sample reservoir into a new collection microfuge tube and centrifuge at 12,000 rpm for 1 minute to collect purified sample.
3.2.7 Dry the sample in a speed vac. This is a STOPPING POINT

3.3 Coupling aa-cDNA to Cy Dye Ester.

3.3.1 Resuspend aminoallyl-labeled cDNA in 7.5 µL 0.075 M sodium carbonate buffer (Na2CO3), pH 9.0 that has been freshly diluted from 1M stock (e.g. 7.5 mL of 1M stock into 92.5 mL water)

3.3.1.1 If using dried-down Cy-dyes in following step, resuspend aa-DNA in 10.0 mL of 0.050 M sodium carbonate buffer, and transfer entire contents to the tube with dried dye pellet. The goal of the coupling reaction is to have approximately 13,000 picomoles of Cy-dye in 50 mM carbonate buffer (final concentration) with your aa-DNA.

 3.3.2 Add 2.5 µL of the appropriate NHS-ester Cy dye (prepared in DMSO: see step 2.5)
Note: To prevent photobleaching of the Cy dyes wrap all reaction tubes in foil and keep them sequestered from light as much as possible using "dark" racks.
3.3.3 Incubate the reaction for 1 hour in the dark at room temperature.

3.4 Reaction Purification II: Removal of uncoupled dye (Qiagen PCR Purification Kit)

3.4.1 To the reaction add 35 µL 100 mM NaOAc pH 5.2 (from freezer).
3.4.2 Add 250 µL (5X reaction volume) Buffer PB (Qiagen supplied).
3.4.3 Place a QIAquick spin column in a 2 mL collection tube (Qiagen supplied), apply the sample to the column, and centrifuge at ~13,000 for 1 minute. Empty collection tube.
3.4.4 To wash, add 0.70 mL Buffer PE (Qiagen supplied) to the column and centrifuge at ~13,000 for 1 minute. Repeat wash step 3.4.4 one time, then proceed to 3.4.5
Note: Make sure Buffer PE has added ethanol before using
3.4.5 Empty collection tube and centrifuge empty column for an additional 1 minute at maximum speed.
3.4.6 Place column in a clean 1.5 mL microfuge tube and carefully add 30 µL Buffer EB (Qiagen supplied) to the center of the column membrane.
3.4.7 Incubate for 1 minute at room temperature.
3.4.8 Elute by centrifugation at ~13,000 rpm for 1 minute.
3.4.9 Elute a second time into the same tube by repeating steps 3.4.6- 3.4.8. The final elution volume should be ~60 µL.
Note: This protocol is modified from the Qiagen QIAquick Spin Handbook (04/2000, pg. 18).

3.5 Analysis of Labeling Reaction

3.5.1 Use a 50 µL Beckman quartz MicroCuvette to analyze the entire undiluted sample in a spectrophotometer.

3.5.1.1 Wash the cuvette with water and blow dry with compressed air duster.
3.5.1.2 Pipette sample into cuvette and place cuvette in spectrophotometer.
3.5.1.3 For each sample measure absorbance at 260 nm and either 550 nm for Cy3 or 650 nm for Cy5, as appropriate.
3.5.1.4 Pipette sample from cuvette back into the original sample tube.
3.5.1.5 For each sample calculate the total picomoles of cDNA synthesized using:

pmol nucleotides = [OD260 * volume (µL) * 37 ng/µL * 1000 pg/ng]
                                                324.5 pg/pmol

Note: 1 OD260 = 37 ng/µL for cDNA; 324.5 pg/pmol average molecular weight of a dNTP)

3.5.1.6 For each sample calculate the total picomoles of dye incorporation (Cy3 or Cy5 accordingly) using:

pmol Cy3 = OD550 * volume (µL)
                            0.15

pmol Cy5 = OD650 * volume (µL)
                           0.25

nucleotides/dye ratio =  pmol cDNA
                                  pmol Cy dye

Note: >200 pmol of dye incorporation per sample and a ratio of less than 50 nucleotides/dye molecules is optimal for hybridizations
3.5.1.7 After analysis mix together the two differentially labeled probes (Cy3 vs. Cy5) which will be hybridized to the same microarray slide.

3.5.2 Using a Nanodrop™ Spectrophotometer remove 1 µL of your labeling reaction and place on nanodrop optical stage.
3.5.2.1 Read sample using microarray software and calculate nucleotide/dye ratio as in steps 3.5.1.6

 3.6 Add blocking agents to achieve a final concentration of:

  0.1 mg/mL Cot-1 DNA (e. g. 0.2 µL of 10mg/ml in 20 µL hyb buffer)
  0.02 mg/mL poly d(A)  (e. g. 0.4 µL of 1.0 mg/mL in 20µL hyb buffer)
  Note : Too much poly d(A) will cause a reduction in fluorescence

3.7 Dry the Cy3/Cy5 probe mixture to completion in a speed vac
3.8 Continue with the ORNL AT Hybridization Protocol to apply the probe to a microarray.

Direct labeling of small amounts of total RNA

Sub-micro Cy3&Cy5 Labeling of RNA for Microarrays #2
Modified to use Genisphere Sub-micro kit
**** CAUTION *** Protocol under construction!

In microfuge tube combine: ("Primer Mix", 10ul)
2ul total RNA (~.5 ug)
3ul RT Primer (0.2 pmole… "Vial 2")
5ul RF-H2O (q.s. 10 ul)
Mix and zing.
Heat to 80°C, 10min, then immediately ice
In separate mfuge tube on ice mix: ("Reaction Mix" 10ul)
4ul 5X RT buffer (Vial 5)
1ul dNTP mix (Vial 4)
4ul RF H20
1ul (200 Units) reverse transcriptase enzyme (Vial 3)
Gently mix and zing.
Add primer mix to reaction mix (final volume 20ul) [primer mix now actually about 7ul]
Mix and incubate at 42°C for 2 hours
Stop reaction with 3.5ul of 0.5M NaOH, 50mM EDTA [Probably should make fresh]
Incubate at 65°C for 10min
Immediately neutralize the mix with 5ml of 1M Tris-HCl, pH= 7.5
· If only using single label, add 38.5ml of 10mM Tris-HCl, pH=8.0, 1mM EDTA (Volume = 67ml)
· For dual labels, combine the Cy3 with the Cy5 (Volume = 57ul). Then rinse Cy3 tube with 10ul of  10mM Tris-pH=8, 1mM EDTA, and combine with label. (Volume = 67ul)
Add the 0.1 ul of 1mg/ml C0T-1 DNA (Volume = 67ul)
Genisphere recommends 20 ug of Linear Polyacrylamide (LPA) (2ul of 10ug/ul) for carrier. (Volume = 69ul)
**************
Concentration via Precipitation:
Add 2 volumes 3M ammonium acetate and mix (140ul)
Add 2.5 volumes of 100% EtOH
Incubate at -20°C 30 minutes
Centrifuge 15 minutes
Discard supe, and wash pellet with 70% EtOH
Centrifuge 5 minutes, discard supe
Dry in Speed-Vac just to the point of pellet (Do not dry completely or it'll be hard to resuspend)
**************
Thaw DNA Hyb buffer (Vial 6) by heating to 65°C with inversion (not vortexing?)
Thaw 3DNA capture reagent (Vial 1) by hand and vortex thoroughly to resuspend aggregates
Resuspend cDNA Samples in 5ml sterile water
Add 2.5ul each of Cy3 and Cy5 3DNA Capture reagents (Vials 1) to cDNA samples
 If using Single channel, use only the one Capture reagent
Add 10ul Hybridization Buffer (Vial 6)
Incubate at 65°C for 15-20 minutes
Add probe mix to microarray and Hyb O/N at 55-65°C.
Washes:
10'@55°C using 2XSSC, 0.2% SDS
10'@RT° using 2XSSC
10'@RT° using 0.2XSSC
Image Arrays 
  
 

GENERAL PREHYBRIDIZATION AND HYBRIDIZATION PROTOCOL

1. MATERIALS:

1.1. Prehybridization Chamber: A slide holder, such as those that are shipped with the new slides is an excellent prehybridization chamber. Up to 5 slides can be prehybridized together using approximately 40mL. A 50mL centrifuge tube also works but can only treat one slide at a time.
1.2. Hybridization chamber: Can be constructed from an empty pipette tip box. Racks must have closed, (one-piece) water-tight bottoms. For detailed instructions on construction

1.2.1. Place pipette tips in front-to-back columns on left and right sides such that two microscope slides will fit next to the small “nubs” sticking up in the middle of the holder “deck”.
1.2.2. Place 20 ml of water into the bottom of the box.
1.2.3. Place the slide with hybridized array is placed onto the deck of the chamber.
1.2.4. Close pipette box lid, and place box inside a food container with a tight-fitting lid, which also has about 20 ml of water in the bottom.
1.2.5. The assembled setup is placed into a 42°C water bath (floating or on a support). A water-jacketed incubator should also work well for hybridizations.
1.2.6. Hybridize 16-18 hours, although hybridization can proceed up to 3 days.

1.3. Coverslips:

1.3.1. Use a 24mm X 24mm HybriSlip (Grace Bio-Labs, HS2424) for 4 X 4 metagrid arrays
1.3.2. Use 22|X25 LifterSlip (Erie Scientific Co., 22|x25-2-4635) for 4 X 4 metagrid arrays

2. REAGENTS:
 

PreHybridization Buffer:	STOCKS	For 50ml use:
25% Deionized formamide	100%	12.5ml
5X SSC	20X	12.5ml
0.1% SDS or Sarcosyl *	10%	0.5 ml
1.0% Bovine Serum Albumin	2.0%	25ml
*(Sarcosyl [N-lauroylsarcosine] is more soluble in high salt, and appeared to improve (lower) background when used in the prehybridization step, but NOT in the hybridization)
Hybridization Buffer:	STOCKS	For 1.0ml use:
25% Deionized formamide	100%	250µL
5X SSC	20X	250µL
0.1% SDS	20%	5µL
Water		495µL

 3. PROCEDURE:

3.1. Prehybridization of cDNA array spotted using 50%DMSO.

3.1.1. Prehybridize the slide in a Slide Holder, in 20-40ml Prehybridization solution (enough to cover the entire array) that has been pre-warmed to 42°C.
3.1.2. Incubate for 1 hour at 42°C
3.1.3. Remove the slide and immediately rinse both sides briefly in gently flowing distilled (Nanopure) water
3.1.4. Blow dry with compressed gas. Alternatively, the slide can be spun dry in a centrifuge. The slide is now ready to hybridize. Store prehybridized slides at room temperature, but use within an hour of completing the prehybridization step.

3.2. Hybridization of labeled RNA to array using coverslips

3.2.1. For a 20X20mm (16-pin) array, using 22X22mm HybriSlips, resuspend sample in 20 ml of Hybridization Buffer
3.2.1.1. HybriSlip Rule: 0.041 X [area of coverslip in mm2] = ml of hybridization buffer
3.2.2. For 22X25mm "Lifter Slips" use 30ml of hybridization solution for 20X20 mm array
3.2.2.1. LifterSlip Rule: 0.054 X [area of coverslip in mm2] = ml of hyb solution)
3.2.2.1.1. Note: LifterSlips should be blown with clean air to remove dust.
3.2.3. Allow labeled samples to sit, protected from light, on ice 10 to 15 minutes, then resuspend again.
3.2.4. Secure the top of the microfuge tube with clip, and heat to 95°C for 5 min (in water) or 10 min (in heat block) to denature. This is critical!
3.2.5. Centrifuge 2 minutes. Do not transfer to ice.
3.2.6. Apply to the microarray, without touching the surface and avoiding bubbles. Place coverslip slowly down onto array. If bubbles form, do not attempt to “push” them out. This will damage your array.
3.2.7. Hybridize the array in a humidified container overnight at 42°C.

3.3. Washing hybridized array

3.3.1. Remove slide from Hybridization chamber avoiding water dripping onto array.
3.3.2. Briefly dip slide into a 50ml tube filled with prewarmed first wash solution, until the coverslips fall away. Agitate gently to prevent the dislodged coverslip from striking the hybridized array.
3.3.3. Wash array in covered (darkened) container  with moderate stirring (no bubbles)

3.3.3.1. First wash: 1 X 5min in 2X SSC, 0.1% SDS @ 42°C (37-42°C is OK)
3.3.3.2. Second washes: 2 X 5min in 0.2X SSC at RT°
3.3.3.3. Final washes: 4 X 1min in 0.1X SSC at RT°

3.3.4. Holding the slide at the bottom, by the barcode, with the arrays upward, rapidly dry with a stream of filtered air or compressed gas blowing towards the barcode at the bottom of the slide.

3.3.4.1. If using compressed “duster” cans, do not shake & hold them 8-10 inches away from slide.

3.3.4.2. Immediately placed slide in a dark dry environment to prevent bleaching of the dye

3.4. Image array 
  
 

Protecting Cy-reactions from light

Cover a closed rack with foil and puch tubes through to produce a light-resistant workrack

ORNL Aminosilane Slide spotting protocols
    Postprocessing 
  
 

ORNL Pre-hybridization station

ORNL Hybridization Chamber (Made from a tip box!)

ORNL High-Throughput Wash Station (Made from histological staining setup)