Probe labeling for Affymetrix microarrays

PROBE LABELING FOR AFFYMETRIX MICROARRAYS
	Last Update: December 2006
	Based on the Affymetrix Technical Manual GeneChip Expression Analysis

One-Cycle cDNA Synthesis

PROCEDURE - Step 1 - Preparation of Poly-A RNA controls for One-Cycle cDNA synthesis (Spike-in controls)
Use Eukaryotic Poly-A RNA Control Kit for this step
NOTE: designed specifically to provide exogenous positive controls to monitor the entire eukaryotic target labeling process, a set of poly-A RNA controls is supplied in the GeneChip Eukaryotic Poly-A RNA Control Kit. Each eukaryotic GeneChip probe array contains probe sets for several B. subtilis genes that are absent in eukaryotic samples (lys, phe, thr, and dap). These poly-A RNA controls are in vitro synthesized, and the polyadenylated transcripts for the B. subtilis genes are pre-mixed at staggered concentrations
1. To prepare the poly-A RNA dilutions for 5 μg of total RNA:
2. Add 2 μL of the Poly-A Control Stock to 38 μL of Poly-A Control Dil Buffer for the First Dilution (1:20)
3. Mix thoroughly and spin down to collect the liquid at the bottom of the tube
4. Add 2 μL of the First Dilution to 98 μL of Poly-A Control Dil Buffer to prepare the Second Dilution (1:50)
5. Mix thoroughly and spin down to collect the liquid at the bottom of the tube
6. Add 2 μL of the Second Dilution to 18 μL of Poly-A Control Dil Buffer to prepare the Third Dilution (1:10)
7. Mix thoroughly and spin down to collect the liquid at the bottom of the tube
8. Add 2 μL of this Third Dilution to 5 μg of sample total RNA
NOTE : The First Dilution of the poly-A RNA controls can be stored up to six weeks in a non-frost-free freezer at -20°C and frozen-thawed up to eight times

PROCEDURE - Step 2 - First-Strand cDNA Synthesis
One-Cycle cDNA Synthesis Kit is used for this step
1. Mix 1-5 μg of RNA sample, 2 μL of diluted poly-A RNA controls, 2 μL of T7-Oligo(dT) Primer, and dH₂O to 12 μL:
1.1 Place total RNA (1 μg to 5 μg) in a 0.2 mL PCR tube
1.2 Add 2 μL of the appropriately diluted poly-A RNA controls (See Step 1: Preparation of Poly-A RNA Controls for One-Cycle cDNA Synthesis (Spike-in Controls)
1.3 Add 2 μL of 50 μM T7-Oligo(dT) Primer
1.4 Add RNase-free Water to a final volume of 12 μL
1.5 Gently flick the tube a few times to mix, and then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
1.6 Incubate the reaction for 10 minutes at 70°C
1.7 Cool the sample at 4°C for at least 2 minutes
1.8 Centrifuge the tube briefly (~5 seconds) to collect the sample at the bottom of the tube
2. In a separate tube, assemble the First-Strand Master Mix
2.1 Prepare sufficient First-Strand Master Mix for all of the RNA samples. When there are more than 2 samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process.
2.2 For a single reaction mix: 4 mL of 5X Strand Reaction Mix, 2 mL of DTT (0.1M), and 1 mL of dNTP (10mM) (final volume 7 mL)
2.3 Mix well by flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the master mix at the bottom of the tube
3. Transfer 7 μL of First-Strand Master Mix to each RNA/T7-Oligo(dT) Primer mix for a final volume of 19 μL. Mix thoroughly by flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube, and immediately place the tubes at 42°C
4. Incubate for 2 minutes at 42°C
5. Add 1 mL of SuperScript II to each RNA sample for a final volume of 20 μL
5.1 Mix thoroughly by flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube, and immediately place the tubes at 42°C
6. Incubate for 1 hour at 42°C; then cool the sample for at least 2 minutes at 4°C
NOTE: Cooling the samples at 4°C is required before proceeding to the next step. Adding the Second-Strand Master Mix directly to solutions that are at 42°C will compromise enzyme activity
6.1 After incubation at 4°C, centrifuge the tube briefly (~5 seconds) to collect the reaction at the bottom of the tube and immediately proceed to Step 3: Second-Strand cDNA Synthesis

PROCEDURE - Step 3 - Second-Strand cDNA Synthesis
1. In a separate tube, assemble Second-Strand Master Mix
NOTE: It is recommended to prepare Second-Strand Master Mix immediately before use
1.1 Prepare sufficient Second-Strand Master Mix for all of the samples. When there are more than 2 samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process.
1.2 For a single reaction mix: 91 mL of RNAse-free water, 30 μL of 5X 2nd Strand Reaction Mix, 3 μL of 10mM dNTPs, 1 μL of E. coli DNA ligase, 4 μL of E. coli DNA polymerase I, and 1 μL of RNAse H (total volume 130 μL)
1.3 Mix well by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the solution at the bottom of the tube
2. Add 130 μL of Second-Strand Master Mix to each first-strand synthesis sample from Step 2: First-Strand cDNA Synthesis for a total volume of 150 μL
2.1 Gently flick the tube a few times to mix, and then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
3. Incubate for 2 hours at 16°C
4. Add 2 μL of T4 DNA Polymerase to each sample and incubate for 5 minutes at 16°C
5. After incubation with T4 DNA Polymerase add 10 μL of EDTA, 0.5M and proceed to Cleanup of Double-Stranded cDNA for both the One-Cycle and Two-Cycle Target Labeling Assays
NOTE: Do not leave the reactions at 4°C for long periods of time

Two-Cycle cDNA Synthesis

PROCEDURE - Step 1 - Preparation of Poly-A RNA Controls for Two-Cycle cDNA Synthesis (Spike-in Controls)
1. To prepare the poly-A RNA dilutions for 10 ng of total RNA:
2. Add 2 μL of the Poly-A Control Stock to 38 μL of Poly-A Control Dil Buffer to prepare the First Dilution (1:20)
3. Mix thoroughly and spin down to collect the liquid at the bottom of the tube
4. Add 2 μL of the First Dilution to 98 μL of Poly-A Control Dil Buffer to prepare the Second Dilution (1:50)
5. Mix thoroughly and spin down to collect the liquid at the bottom of the tube
6. Add 2 μL of the Second Dilution to 98 μL of Poly-A Control Dil Buffer to prepare the Third Dilution (1:50)
7. Mix thoroughly and spin down to collect the liquid at the bottom of the tube
8. Add 2 μL of the Third Dilution to 18 μL of Poly-A Control Dil Buffer to prepare the Fourth Dilution (1:10)
9. Use the Fourth Dilution to prepare the solution described next
NOTE: the first dilution of the poly-A RNA controls (1:20) can be stored in a non-frost-free freezer at -20°C up to six weeks and frozen-thawed up to eight times.
Preparation of T7-Oligo(dT) Primer/Poly-A Controls Mix:
1. Prepare a fresh dilution of the T7-Oligo(dT) Primer from 50 μM to 5 μM. The diluted poly-A RNA controls should be added to the concentrated T7-Oligo(dT) Primer as follows, using a non-stick RNase-free microfuge tube.
2. The following recipe is sufficient for 10 samples: 2 mL of 50 mM T7-Oligo(dT) Primer, 2 mL of Diluted Poly-A RNA controls, 16 mL of RNAse-Free water (total volume 20 mL)

PROCEDURE - Step 2 - First-Cycle, First-Strand cDNA Synthesis
Two-Cycle cDNA Synthesis Kit is used for this step
1. Mix total RNA sample and the T7-Oligo(dT) Primer/Poly-A Controls Mix
1.1 Place total RNA sample (10 to 100 ng) in a 0.2 mL PCR tube
1.2 Add 2 μL of the T7-Oligo(dT) Primer/Poly-A Controls Mix (See Step 1: Preparation of Poly-A RNA Controls for Two-Cycle cDNA Synthesis (Spike-in Controls)
1.3 Add RNase-free Water to a final volume of 5 μL
1.4 Gently flick the tube a few times to mix, then centrifuge the tubes briefly (~5 seconds) to collect the solution at the bottom of the tube
1.5 Incubate for 6 minutes at 70°C
1.6 Cool the sample at 4°C for at least 2 minutes. Centrifuge briefly (~5 seconds) to collect the sample at the bottom of the tube
2. In a separate tube, assemble the First-Cycle, First-Strand Master Mix
2.1 Prepare sufficient First-Cycle, First-Strand Master Mix for all of the total RNA samples. When there are more than 2 samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process.
2.2 For a single reaction mix: 2 mL of 5X 1st Strand Reaction Mix, 1 mL of 0.1M DTT, 0.5 mL of RNase Inhibitor, 0.5 mL of 10mM dNTP, and 1 mL of SuperScript II (final volume 5 mL)
2.3 Mix well by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the solution at the bottom of the tube
3. Transfer 5 μL of First-Cycle, First-Strand Master Mix to each total RNA sample/ T7-Oligo(dT) Primer/Poly-A Controls Mix from the previous step for a final volume of 10 μL
3.1 Mix thoroughly by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube, and immediately place the tubes at 42°C
4. Incubate for 1 hour at 42°C
5. Heat the sample at 70°C for 10 minutes to inactivate the RT enzyme, then cool the sample for at least 2 minutes at 4°C
5.1 After the 2 minute incubation at 4°C, centrifuge the tube briefly (~5 seconds) to collect the reaction at the bottom of the tube and immediately proceed to Step 3: First-Cycle, Second-Strand cDNA Synthesis
NOTE: Cooling the sample at 4°C is required before proceeding to the next step. Adding the First-Cycle, Second-Strand Master Mix directly to solutions that are at 70°C will compromise enzyme activity

PROCEDURE - Step 3 - First-Cycle, Second-Strand cDNA Synthesis
Two-Cycle cDNA Synthesis Kit is used for this step
1. In a separate tube, assemble the First-Cycle, Second-Strand Master Mix
NOTE: It is recommended to prepare this First-Cycle, Second-Strand Master Mix immediately before use. Prepare this First-Cycle, Second-Strand Master Mix for at least 4 reactions at one time for easier and more accurate pipetting
1.1 Prepare sufficient First-Cycle, Second-Strand Master Mix for all samples. When there are more than 2 samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process
1.2 For a single reaction mix: 4.8 mL of RNase-free Water, 4 mL of 17.5mM Freshly diluted MgCl₂, 0.4 mL of 10mM dNTP, 0.6 mL of E. coli DNA Polymerase I, 0.2 mL of RNase H (total volume of 10 mL)
1.3 Mix well by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the solution at the bottom of the tube
2. Add 10 μL of the First-Cycle, Second-Strand Master Mix to each sample from Step 2: First-Cycle, First-Strand cDNA Synthesis reaction for a total volume of 20 μL
2.1 Gently flick the tube a few times to mix, and then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
3. Incubate for 2 hours at 16°C, then 10 minutes at 75°C and cool the sample at least 2 minutes at 4°C. Turn the heated lid function off only for the 16°C incubation
3.1 After the 2 minute incubation at 4°C, centrifuge the tube briefly (~5 seconds) to collect the reaction at the bottom of the tube. Proceed to Step 4: First-Cycle, IVT Amplification of cRNA
NOTE: No cDNA cleanup is required at this step

PROCEDURE - Step 4 - First-Cycle, IVT Amplification of cRNA
MEGAscript® T7 Kit (Ambion, Inc.) is used for this step
1. In a separate tube, assemble the First-Cycle, IVT Master Mix at room temperature
1.1 Prepare sufficient First-Cycle, IVT Master Mix for all of the samples. When there are more than 2 samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process
1.2 For a single reaction mix: 5 mL of 10X Reaction Buffer, 5 mL of ATP solution, 5 mL of CTP solution, 5 mL of UTP solution, 5 mL of GTP solution, and 5 mL of Enzyme mix (final volume of 30 mL)
1.3 Mix well by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the solution at the bottom of the tube
2. Transfer 30 μL of First-Cycle, IVT Master Mix to each cDNA sample
2.1 At room temperature, add 30 μL of the First-Cycle, IVT Master Mix to each 20 μL of cDNA sample from Step 3: First-Cycle, Second-Strand cDNA Synthesis for a final volume of 50 μL
2.2 Gently flick the tube a few times to mix, then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
3. Incubate for 16 hours at 37°C
3.1 After the 16 hour incubation at 37°C, centrifuge the tube briefly (~5 seconds) to collect the reaction at the bottom of the tube
3.2 The sample is now ready to be purified in Step 5: First-Cycle, Cleanup of cRNA. Alternatively, samples may be stored at -20°C for later use.

PROCEDURE - Step 5 - First-Cycle, Cleanup of cRNA
Sample Cleanup Module is used for this step
NOTE: IVT cRNA Wash Buffer is supplied as a concentrate. Before using for the first time, add 20 mL of ethanol (96-100%), as indicated on the bottle, to obtain a working solution, and checkmark the box on the left-hand side of the bottle label to avoid confusion
NOTE: IVT cRNA Binding Buffer may form a precipitate upon storage. If necessary, redissolve by warming in a water bath at 30°C, and then place the buffer at room temperature
NOTE: All steps of the protocol should be performed at room temperature. During the procedure, work without interruption
1. Add 50 μL of RNase-free Water to the IVT reaction and mix by vortexing for 3 seconds
2. Add 350 μL IVT cRNA Binding Buffer to the sample and mix by vortexing for 3 seconds
3. Add 250 μL ethanol (96-100%) to the lysate, and mix well by pipetting. Do not centrifuge
4. Apply sample (700 μL) to the IVT cRNA Cleanup Spin Column sitting in a 2 mL Collection Tube. Centrifuge for 15 seconds at ≥ 8,000 x g (≥ 10,000 rpm). Discard flow-through and Collection Tube
5. Transfer the spin column into a new 2 mL Collection Tube (supplied). Pipet 500 μL IVT cRNA Wash Buffer onto the spin column. Centrifuge for 15 seconds at ≥ 8,000 x g (≥ 10,000 rpm) to wash. Discard flow-through
NOTE: IVT cRNA Wash Buffer is supplied as a concentrate. Ensure that ethanol is added to the IVT cRNA Wash Buffer before use (see IMPORTANT note above before starting)
6. Pipet 500 μL 80% (v/v) ethanol onto the spin column and centrifuge for 15 seconds at ≥ 8,000 x g (≥ 10,000 rpm). Discard flow-through
7. Open the cap of the spin column and centrifuge for 5 minutes at maximum speed (≤ 25,000 x g). Discard flow-through and Collection Tube
7.1 Place columns into the centrifuge using every second bucket. Position caps over the adjoining bucket so that they are oriented in the opposite direction to the rotation (i.e., if the microcentrifuge rotates in a clockwise direction, orient the caps in a counterclockwise direction). This avoids damage of the caps
NOTE: Label the collection tubes with the sample name. During centrifugation some column caps may break, resulting in loss of sample information
NOTE: Centrifugation with open caps allows complete drying of the membrane
8. Transfer spin column into a new 1.5 mL Collection Tube (supplied), and pipet 13 μL of RNase-free Water directly onto the spin column membrane. Ensure that the water is dispensed directly onto the membrane. Centrifuge 1 minute at maximum speed (≤ 25,000 x g) to elute. The average volume of eluate is 11 μL from 13 μL RNase-free Water
9. To determine cRNA yield for samples starting with 50 ng or higher, remove 2 μL of the cRNA, and add 78 μL of water to measure the absorbance at 260 nm. Use 600 ng of cRNA in the following Step 6: Second-Cycle, First-Strand cDNA Synthesis Reaction. For starting material less than 50 ng, or if the yield is less than 600 ng, use the entire eluate for the Second-Cycle, First-Strand cDNA Synthesis Reaction
9.1 Samples can be stored at -20°C for later use, or proceed to Step 6: Second-Cycle, First- Strand cDNA Synthesis

PROCEDURE - Step 6 - Second-Cycle, First-Strand cDNA Synthesis
Two-Cycle cDNA Synthesis Kit is used for this step
1. Mix cRNA and diluted random primers
1.1 Make a fresh dilution of the Random Primers (final concentration 0.2 μg/μL). Mix 2μL of Random Primers, 3 μg/μL, with 28 μL RNase-free Water
1.2 Add 2 μL of diluted random primers to purified cRNA from Step 5: First-Cycle, Cleanup of cRNA, substep9 on page2.1.27 and add RNase-free Water for a final volume of 11μL
1.3 Incubate for 10 minutes at 70°C
1.4 Cool the sample at 4°C for at least 2 minutes. Centrifuge briefly (~5 seconds) to collect the sample at the bottom of the tube
2. In a separate tube, assemble the Second-Cycle, First-Strand Master Mix
2.1 Prepare sufficient Second-Cycle, First-Strand Master Mix for all of the samples. When there are more than two samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process.
2.2 For a single reaction mix: 4 mL of 5X 1st Strand Reaction Mix, 2 mL of 0.1M DTT, 1 mL of RNase Inhibitor, 1 mL of 10mM dNTP, and 1 mL of SuperScript II (Total volume of 9 mL)
2.3 Mix well by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the solution at the bottom of the tube
3. Transfer 9 μL of Second-Cycle, First-Strand Master Mix to each cRNA/random primer sample from Step 6: Second-Cycle, First-Strand cDNA Synthesis, for a final volume of 20 μL
3.1 Mix thoroughly by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube and place the tubes at 42°C immediately
4. Incubate for 1 hour at 42°C, then cool the sample for at least 2 minutes at 4°C. After the incubation at 4°C, centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
5. Add 1 μL of RNase H to each sample for a final volume of 21 μL. Mix thoroughly by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube and incubate for 20 minutes at 37°C
6. Heat the sample at 95°C for 5 minutes. Cool the sample for at least 2 minutes at 4°C; then, proceed directly to Step 7: Second-Cycle, Second-Strand cDNA Synthesis

PROCEDURE - Step 7 - Second-Cycle, Second-Strand cDNA Synthesis
Two-Cycle cDNA Synthesis Kit is used for this step
1. Add 4 μL of diluted T7-Oligo(dT) Primer to each sample
1.1 Make a fresh dilution of the T7-Oligo(dT) Primer (final concentration 5 μM). Mix 2μL of T7-Oligo(dT) Primer, 50 μM, with 18 μL of RNase-free Water
1.2 Add 4 μL of diluted T7-Oligo(dT) Primer to the sample from Step 6: Second-Cycle, First-Strand cDNA Synthesis for a final volume of 25 μL
1.3 Gently flick the tube a few times to mix, and then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
1.4 Incubate for 6 minutes at 70°C
1.5 Cool the sample at 4°C for at least 2 minutes. Centrifuge briefly (~5 seconds) to collect sample at the bottom of the tube
NOTE: cooling the samples at 4°C is required before proceeding to the next step. Adding the Second-Strand Master Mix directly to solutions that are at 70°C will compromise enzyme activity
NOTE: it is recommended to prepare the Second-Cycle, Second-Strand Master Mix immediately before use.
2. In a separate tube, assemble the Second-Cycle, Second-Strand Master Mix
2.1 Prepare sufficient Second-Cycle, Second-Strand Master Mix for all of the samples. When there are more than two samples, it is prudent to include additional material to compensate for potential pipetting inaccuracy or solution lost during the process
2.2 For a single reaction mix: 88 mL of RNase-free Water, 30 mL of 5X 2nd Strand Reaction Mix, 3 mL of 10mM dNTP, and 4 mL of E. coli DNA Polymerase I (Total volume of 125 mL)
2.3 Mix well by gently flicking the tube a few times. Centrifuge briefly (~5 seconds) to collect the master mix at the bottom of the tube
3. Add 125 μL of the Second-Cycle, Second-Strand Master Mix to each sample from Step 7: Second-Cycle, Second-Strand cDNA Synthesis, for a total volume of 150 μL
3.1 Gently flick the tube a few times to mix, then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of tube
4. Incubate for 2 hours at 16°C
5. Add 2 μL of T4 DNA Polymerase to the samples for a final volume of 152 μL. Gently flick the tube a few times to mix, and then centrifuge briefly (~5 seconds) to collect the reaction at the bottom of the tube
6. Incubate for 10 minutes at 16°C, then cool the sample at 4°C for at least 2 minutes. Centrifuge briefly (~5 seconds) to collect sample at the bottom of the tube. After the incubation at 4°C, centrifuge the tube briefly (~5 seconds) to collect the reaction at the bottom of the tube. Proceed to Cleanup of Double-Stranded cDNA for Both the One-Cycle and Two-Cycle Target Labeling Assays. Alternatively, immediately freeze the sample at –20°C for later use. Do not leave the reaction at 4°C for long periods of time

Cleanup of Double-Stranded cDNA for Both the One-Cycle and Two-Cycle Target Labeling Assays

Sample Cleanup Module is used for cleaning up the double-stranded cDNA
NOTE: cDNA Wash Buffer is supplied as a concentrate. Before using for the first time, add 24mL of ethanol (96-100%), as indicated on the bottle, to obtain a working solution, and checkmark the box on the left-hand side of the bottle label to avoid confusion
NOTE: All steps of the protocol should be performed at room temperature. During the procedure, work without interruption
NOTE: If cDNA synthesis was performed in a reaction tube smaller than 1.5 mL, transfer the reaction mixture into a 1.5 or 2 mL microfuge tube (not supplied) prior to addition of cDNA Binding Buffer
1. Add 600 μL of cDNA Binding Buffer to the double-stranded cDNA synthesis preparation. Mix by vortexing for 3 seconds
2. Check that the color of the mixture is yellow (similar to cDNA Binding Buffer without the cDNA synthesis reaction)
NOTE: If the color of the mixture is orange or violet, add 10 μL of 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow
3. Apply 500 μL of the sample to the cDNA Cleanup Spin Column sitting in a 2 mL Collection Tube (supplied), and centrifuge for 1 minute at ≥ 8,000 x g (≥ 10,000 rpm). Discard flow-through
4. Reload the spin column with the remaining mixture and centrifuge as above. Discard flow-through and Collection Tube
5. Transfer spin column into a new 2 mL Collection Tube (supplied). Pipet 750 μL of the cDNA Wash Buffer onto the spin column. Centrifuge for 1 minute at ≥ 8,000 x g (≥ 10,000 rpm). Discard flow-through
NOTE: cDNA Wash Buffer is supplied as a concentrate. Ensure that ethanol is added to the cDNA Wash Buffer before use (see IMPORTANT note above before starting)
6. Open the cap of the spin column and centrifuge for 5 minutes at maximum speed (≤ 25,000 x g). Discard flow-through and Collection Tube
NOTE: Label the collection tubes with the sample name. During centrifugation some column caps may break, resulting in loss of sample information
6.1 Place columns into the centrifuge using every second bucket. Position caps over the adjoining bucket so that they are oriented in the opposite direction to the rotation (i.e., if the microcentrifuge rotates in a clockwise direction, orient the caps in a counterclockwise direction). This avoids damage of the caps
NOTE: Centrifugation with open caps allows complete drying of the membrane
7. Transfer spin column into a 1.5 mL Collection Tube, and pipet 14 μL of cDNA Elution Buffer directly onto the spin column membrane. Incubate for 1 minute at room temperature and centrifuge 1 minute at maximum speed (≤ 25,000 x g) to elute. Ensure that the cDNA Elution Buffer is dispensed directly onto the membrane. The average volume of eluate is 12 μL from 14 μL Elution Buffer
NOTE: We do not recommend RNase treatment of the cDNA prior to the in vitro transcription and labeling reaction; the carry-over ribosomal RNA does not seem to inhibit the reaction
NOTE: We do not recommend gel analysis or spectrophotometric quantitation for cDNA prepared from total RNA. This is due to the presence of other nucleic acid species in the sample that can interfere with the results
7.1 After cleanup, please proceed to Synthesis of Biotin-Labeled cRNA for Both the One-Cycle and Two-Cycle Target Labeling Assays

Synthesis of Biotin-Labeled cRNA for Both the One-Cycle and Two-Cycle Target Labeling Assays

GeneChip IVT Labeling Kit is used for this step
NOTE: This kit is only used for the IVT labeling step for generating biotin-labeled cRNA. For the IVT amplification step using unlabeled ribonucleotides in the First Cycle of the Two-Cycle cDNA Synthesis Procedure, a separate kit is recommended (MEGAscript® T7 Kit, Ambion, Inc.). Use only nuclease-free water, buffers, and pipette tips
NOTE: Store all reagents in a -20°C freezer that is not self-defrosting. Prior to use, centrifuge all reagents briefly to ensure that the solution is collected at the bottom of the tube. The Target Hybridizations and Array Washing protocols have been optimized specifically for this IVT Labeling Protocol. Closely follow the recommendations described below for maximum array performance
1. Use all the cDNA made from 1-5 mg of RNA for each IVT reaction following the cDNA cleanup step
2. Transfer the needed amount of template cDNA to RNase-free microfuge tubes and add the following reaction components in the order indicated in the table below. If more than one IVT reaction is to be performed, a master mix can be prepared by multiplying the reagent volumes by the number of reactions. Do not assemble the reaction on ice, since spermidine in the 10X IVT Labeling Buffer can lead to precipitation of the template cDNA
2.1 Set up reaction: template cDNA (from 1-5 mg of RNA), 4 mL of 10X IVT Labelling Buffer, 12 mL of IVT Labeling NTP Mix, 4 mL of Labeling Enzyme Mix, and add RNase-free Water to 40 mL
3. Carefully mix the reagents and collect the mixture at the bottom of the tube by brief (~5 seconds) microcentrifugation
4. Incubate at 37°C for 16 hours. To prevent condensation that may result from water bath-style incubators, incubations are best performed in oven incubators for even temperature distribution, or in a thermal cycler
NOTE: Overnight IVT reaction time has been shown to maximize the labeled cRNA yield with high-quality array results. Alternatively, if a shorter incubation time (4 hours) is desired, 1 μL (200 units) of cloned T7 RNA polymerase (can be purchased directly from Ambion, P/N 2085) can be added to each reaction and has been shown to produce adequate labeled cRNA yield within 4 hours. The two different incubation protocols generate comparable array results, and users are encouraged to choose the procedure that best fits their experimental schedule and process flow
5. Store labeled cRNA at -20°C, or -70°C if not purifying immediately. Alternatively, proceed to Cleanup and Quantification of Biotin-Labeled cRNA

Cleanup and Quantification of Biotin-Labeled cRNA

Sample Cleanup Module is used for cleaning up the biotin-labeled cRNA

PROCEDURE - Step 1 - Cleanup of Biotin-Labeled cRNA
NOTE: It is essential to remove unincorporated NTPs, so that the concentration and purity of cRNA can be accurately determined by 260 nm absorbance
NOTE: DO NOT extract biotin-labeled RNA with phenol-chloroform. The biotin will cause some of the RNA to partition into the organic phase. This will result in low yields
NOTE: Save an aliquot of the unpurified IVT product for analysis by gel electrophoresis
NOTE: IVT cRNA Wash Buffer is supplied as a concentrate. Before using for the first time, add 20 mL of ethanol (96-100%), as indicated on the bottle, to obtain a working solution, and checkmark the box on the left-hand side of the bottle label to avoid confusion
NOTE: IVT cRNA Binding Buffer may form a precipitate upon storage. If necessary, redissolve by warming in a water bath at 30°C, and then place the buffer at room temperature
NOTE: All steps of the protocol should be performed at room temperature. During the procedure, work without interruption
1. Add 60 μL of RNase-free Water to the IVT reaction and mix by vortexing for 3 seconds
2. Add 350 μL IVT cRNA Binding Buffer to the sample and mix by vortexing for 3 seconds
3. Add 250 μL ethanol (96-100%) to the lysate, and mix well by pipetting. Do not centrifuge
4. Apply sample (700 μL) to the IVT cRNA Cleanup Spin Column sitting in a 2 mL Collection Tube. Centrifuge for 15 seconds at ≥ 8,000 x g (≥ 10,000 rpm). Discard flow-through and Collection Tube
5. Transfer the spin column into a new 2 mL Collection Tube (supplied). Pipet 500 μL IVT cRNA Wash Buffer onto the spin column. Centrifuge for 15seconds at ≥ 8,000 x g (≥ 10,000 rpm) to wash. Discard flow-through
NOTE: IVT cRNA Wash Buffer is supplied as a concentrate. Ensure that ethanol is added to the IVT cRNA Wash Buffer before use (see IMPORTANT note above before starting).
6. Pipet 500 μL 80% (v/v) ethanol onto the spin column and centrifuge for 15 seconds at ≥ 8,000 x g (≥ 10,000 rpm). Discard flow-through
7. Open the cap of the spin column and centrifuge for 5 minutes at maximum speed (≤ 25,000 x g). Discard flow-through and Collection Tube
7.1 Place columns into the centrifuge using every second bucket. Position caps over the adjoining bucket so that they are oriented in the opposite direction to the rotation (i.e., if the microcentrifuge rotates in a clockwise direction, orient the caps in a counterclockwise direction). This avoids damage of the caps
NOTE: Label the collection tubes with the sample name. During centrifugation some column caps may break, resulting in loss of sample information
NOTE: Centrifugation with open caps allows complete drying of the membrane
8. Transfer spin column into a new 1.5 mL Collection Tube (supplied), and pipet 11 μL of RNase-free Water directly onto the spin column membrane. Ensure that the water is dispensed directly onto the membrane. Centrifuge 1 minute at maximum speed (≤ 25,000 x g) to elute
9. Pipet 10 μL of RNase-free Water directly onto the spin column membrane. Ensure that the water is dispensed directly onto the membrane. Centrifuge 1 minute at maximum speed (≤ 25,000 x g) to elute
9.1 For subsequent photometric quantification of the purified cRNA, we recommend dilution of the eluate between 1:100 fold and 1:200 fold
9.2 Store cRNA at -20°C, or -70°C if not quantitated immediately. Alternatively, proceed to Step 2: Quantification of the cRNA

PROCEDURE - Step 2 - Quantification of the cRNA
1. Use spectrophotometric analysis to determine the cRNA yield. Apply the convention that 1absorbance unit at 260 nm equals 40 μg/mL RNA
2. Check the absorbance at 260 nm and 280 nm to determine sample concentration and purity
3. Maintain the A₂₆₀/A₂₈₀ ratio close to 2.0 for pure RNA (ratios between 1.9 and 2.1 are acceptable)
4. For quantification of cRNA when using total RNA as starting material, an adjusted cRNA yield must be calculated to reflect carryover of unlabeled total RNA. Using an estimate of 100% carryover, use the formula below to determine adjusted cRNA yield:
4.1 adjusted cRNA yield = RNA_m - (total RNA_i) (y)
4.2 RNA_m = amount of cRNA measured after IVT (μg)
4.3 total RNA_i = starting amount of total RNA (μg)
4.4 y = fraction of cDNA reaction used in IVT
Example: Starting with 10 μg total RNA, 50% of the cDNA reaction is added to the IVT, giving a yield of 50 μg cRNA. Therefore, adjusted cRNA yield = 50 μg cRNA - (10 μg total RNA) (0.5 cDNA reaction) = 45.0 μg
5. Use adjusted yield in Fragmenting the cRNA for Target Preparation

PROCEDURE - Step 3 - Checking Unfragmented Samples by Gel Electrophoresis
Gel electrophoresis of the IVT product is done to estimate the yield and size distribution of labeled transcripts. The following are examples of typical cRNA products examined on an Agilent 2100 Bioanalyzer

Fragmenting the cRNA for Target Preparation

Sample Cleanup Module is used for this step
NOTE: Fragmentation of cRNA target before hybridization onto GeneChip probe arrays has been shown to be critical in obtaining optimal assay sensitivity
NOTE: Affymetrix recommends that the cRNA used in the fragmentation procedure be sufficiently concentrated to maintain a small volume during the procedure. This will minimize the amount of magnesium in the final hybridization cocktail. Fragment an appropriate amount of cRNA for hybridization cocktail preparation and gel analysis
1. The Fragmentation Buffer has been optimized to break down full-length cRNA to 35 to 200 base fragments by metal-induced hydrolysis
2. Set up a fragmentation reaction mix for cRNA samples at a final concentration of 0.5 μg/μL. Use adjusted cRNA concentration, as described in Step 2: Quantification of the cRNA. The total volume of the reaction may be scaled up or down dependent on the amount of cRNA to be fragmented
2.1 Set up reaction: Use 20 mg of cRNA (1 to 21 mL) , 8 mL of 5X Fragmentation Buffer, and RNase-free Water to a final volume of 40 mL
3. Incubate at 94°C for 35 minutes. Put on ice following the incubation
4. Save an aliquot for analysis on the Bioanalyzer
4.1 The standard fragmentation procedure should produce a distribution of RNA fragment sizes from approximately 35 to 200 bases
5. Store undiluted, fragmented sample cRNA at -20°C (or -70°C for longer-term storage) until ready to perform the hybridization
The probe is now ready for hybridization (Affymetrix Microarray Target Hybridization)