Labeling Hybridization Protocol for Affymetrix Probe Arrays

General Instructions

1. The following protocol is an abbreviated version of the protocol in the Affymetrix Gene Expression Analysis Technical manual.
2. Read through the manual before following the steps below.
3. Use only RNase/DNase free reagents/supplies.
4. Wear gloves at all times.

Synthesis of Double Stranded cDNA from Total RNA

(Using the Supercript Choice System by Gibco)

Use a starting amount of 5.0 – 40.0 µg total RNA.

First Strand cDNA Synthesis (using 5.0 µg of total RNA)

1. In a 1.5-mL microcentrifuge tube, combine the following: x µL of total RNA, 12- x µL of RNase/DNase free water and 1 µL of T7- (dT)24 primer.
2. Incubate at 70°C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
3. Add the following to the tube: 4 µL of 5X First strand cDNA buffer, 2 µL of 0.1 M DTT and 1 µL of 10 mM dNTP mix.
4. Incubate at 42°C for 2 minutes.
5. Add to the tube 1 µL of Superscript II reverse transcriptase.
6. Incubate at 42°C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.

Second Strand cDNA Synthesis (using 5.0 µg of total RNA)

1. Add the following to the First Strand Synthesis tube: 91 µL of RNase/DNase free water, 30 µL of 5X second strand reaction buffer, 3 µL of 10 mM dNTP mix, 1 µL of 10 U/µL DNA Ligase (E. coli), 4 µL of 10 U/µL DNA Polymerase I (E. coli) and 1 µL of 2 U/µL RNase H (E. coli).
2. Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
3. Add 2 µL [10 U] T4 DNA Polymerase.
4. Incubate at 16°C for 5 minutes.
5. Add 10 µL 0.5 M EDTA.

Cleanup of Double-Stranded cDNA

Phenol/Chloroform Extraction

1. Add 162 µL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
2. Centrifuge sample at maximum speed for 2 minutes. Transfer aqueous phase to fresh 1.5-mL microcentrifuge tube.

Ethanol Precipitation

1. Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of absolute ethanol to sample. Vortex.
2. Immediately spin at maximum speed for 20 minutes at room temperature.
3. Remove supernatant. Wash pellet with 500 µL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
4. Remove 80% ethanol. Wash again with 500 µL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
5. Air dry pellet.
6. Resuspend in 12 µL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 µL aliquot to run on gel later.

Synthesis of Biotin-Labeled cRNA (Using in Vitro Transcription Reaction)

IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)

1. Add the following to a fresh 1.5-mL microcentrifuge tube: x µL to give 1 µg double-stranded cDNA, 22 – x µL of RNase/Dnase free water, 4 µL of 10X HY reaction buffer, 4 µL of 10X Biotin-labeled ribonucleotides, 4 µL of 10X DTT, 4 µL of 10X RNase inhibitor mix and 2 µL of T7 RNA polymerase.
2. Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.

IVT Cleanup

• The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
• The RNA elution step is performed twice to increase recovery.
• Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 µl of water.

Quantifying the cRNA

1. Prepare 1:50 dilution of the RNA to a total of 100 µl.
2. Check OD at 260 nm and 280 nm to determine sample concentration and purity. A₂₆₀/A₂₈₀ ratio between 1.9 and 2.1 is acceptable.
3. The following formula is used to determine the cRNA yield.
   
   Adjusted cRNA yield=RNA* - (total RNA#)($)
   RNA* = amount of cRNA measured after IVT (µg)
   total RNA# = sterting amount of total RNA (µg)
   $ = fraction of cRNA reaction used in IVT

cRNA Fragmentation

1. To 20 µg of unadjusted cRNA 2 µl of 5X fragmentation buffer is added for every 8 µl of RNA, and the total volume is adjusted to 40 µl with water.
2. The fragmentation reaction is performed at 94°C for 35 minutes and kept on ice post incubation.
3. An aliquot of cRNA is saved for gel analysis and the rest is stored at –20°C until ready to perform hybridization.

Gel Electrophoresis

1. Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
2. Mix RNA with loading dye and heat to 65°C for 5 minutes before loading on the gel.

Hybridization

1. Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65°C for 5 minutes.
2. Vortex the vials to ensure complete mixing and then briefly spin down the samples.
3. Prepare the hybridization cocktail as follows:
   

   Reagents	Mini Array	Midi Array	Standard Array
   Fragmented cRNA	5 µg	10 µg	15 µg
   Control oligo B2	1.7 µl	3.3 µl	5 µl
   20X eukaryotic hybridization controls	5 µl	10 µl	15 µl
   Herring sperm DNA	1 µl	2 µl	3 µl
   Acetylated BSA	1 µl	2 µl	3 µl
   2X Hybridization buffer	50 µl	100 µl	150 µl
   Water	to 100 µl	to 200 µl	to 300 µl

   
   
4. Equilibrate probe array to room temperature.
5. Heat the hybridization cocktail to 99°C for 5 minutes, spin briefly and transfer to 45°C for 5 minutes.
6. Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
7. Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45°C for 10 minutes with rotation.
8. Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45°C with rotation.

Washing and Staining Probe Arrays

Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.