Affymetrix GeneChip

Affymetrix revises their protocols and assays from time to time. For the most accurate information please check their website.

Affymetrix GeneChip Materials /Reagents

cDNA Synthesis/Purification

  • T7-Oligo(dT) – 50µM, HPLC purified
  • SuperScript II Reverse Transcriptase
  • E. coli DNA ligase
  • E. coli DNA Polymerase I
  • E. coli RNase H
  • T4 DNA Polymerase
  • 5X Second strand buffer
  • 10mM dNTP mix
  • GeneChip Sample Cleanup Module

Synthesis of Biotin-Labeled cRNA/Purification

  • ENZO BioArray RNA Transcript Labeling Kit or Affymetrix IVT Labeling Kit
  • GeneChip Sample Cleanup Module

Miscellaneous Reagents/Supplies

  • 100% ethanol – room temperature
  • 80%   ethanol – room temperature
  • Water, Molecular Biology Grade or DEPC-treated
  • Ethidium Bromide
  • Sterile, RNase-free, microcentrifuge tubes, 1.5mL
  • Micropipettors
  • Sterile-barrier, RNase-free pipette tips
  • Mini agarose gel electrophoresis unit with appropriate buffers
  • UV Spectrophotometer
  • Cooling water bath

Hybridization

  • Water, Molecular Biology Grade or DEPC-treated
  • Acetylated Bovine Serum Albumin (BSA) solution (50 mg/mL)
  • Herring Sperm DNA
  • GeneChip Eukaryotic Hybridization Control Kit
  • 5M NaCl, RNase-free, DNase-free
  • MES Free Acid Monohydrate
  • MES Sodium Salt
  • EDTA Disodium Salt

Miscellaneous Reagents/Supplies

  • Surfact-Amps 20 (Tween-20), 10%
  • Hybridization Oven 640
  • Sterile, RNase-free, microcentrifuge tubes, 1.5mL
  • Micropipettors
  • Sterile-barrier pipette tips and non-barrier tips
  • Heatblock

Washing, Staining, and Scanning

  • Water, Molecular Biology Grade
  • Distilled water
  • Acetylated Bovine Serum Albumin (BSA) solution (50 mg/mL)
  • R-Phycoerythrin Streptavidin
  • 20X SSPE (3M NaCl, 0.2M NaH2PO4, 0.02M EDTA)
  • Goat IgG, Reagent Grade
  • Anti-streptavidin antibody (goat), biotinylated
  • 10% surfact-Amps 20 (Tween-20)

Reagent/Buffer Preparation

12X MES Stock (1.22M MES, 0.89M [Na+]), 1000mL:

  • 70.4g MES-free acid monohydrate
  • 193.3g MES Sodium Salt
  • 800mL water, Molecular Biology Grade
  • Mix and adjust volume to 1000mL with water
  • pH should be between 6.5 and 6.7.
  • Filter through a 0.2µm filter.
  • Do not autoclave.  Store at 4°C.  Shield from light.  Discard solution if yellow.

2X Hybridization Buffer (final concentration is 100mM MES, 1M [Na+], 20mM EDTA,0.01% Tween-20), 50mL:

  • 8.3mL of 12X MES Stock
  • 17.7mL of 5M NaCl
  • 4.0mL of 0.5M EDTA
  • 0.1mL of 10% Tween-20
  • 19.9mL of water, Molecular Biology Grade
  • Store at 4°C.  Shield from light.

Wash A:  Non-Stringent Wash Buffer (6X SSPE, 0.01% Tween-20), 1000mL:

  • 300mL of 20X SSPE
  • 1.0mL of 10% Tween-20
  • 699mL of water, Molecular Biology Grade
  • Filter through a 0.2µm filter

Wash B:  Stringent Wash Buffer (100mM MES, 0.1M [Na+], 0.01% Tween-20), 1000mL:

  • 83.3mL of 12X MES Stock Buffer
  • 5.2mL of 5M NaCl
  • 1.0mL of 10% Tween-20
  • 910.5mL of water, Molecular Biology Grade
  • Filter through a 0.2µm filter
  • Store at 4°C.  Shield from light.

2X Stain Buffer (2X MES Stain Buffer) (Final 1X conc.:  100mM MES, 1M [Na+], 0.05% Tween-20), 250mL:

  • 41.7mL 12X MES Stock Buffer
  • 92.5mL 5M NaCl
  • 2.5mL 10% Tween-20
  • 113.3mL of water, Molecular Biology Grade
  • Filter through a 0.2µm filter.
  • Store at 4°C.  Shield from light.

10mg/mL Goat IgG Stock:

  • Resuspend 50mg in 5mL of 150mM NaCl
  • Store at 4°C.

 

Affymetrix GeneChip Protocol

Day #1

cDNA Synthesis

◊ Starting amount of high-purity, total RNA is 5µg.  Total cDNA synthesis volume is 20µl.  Reaction components total 10µl.  Volume of RNA sample will vary based upon measured concentration.  Water is added to reach a final reaction volume of 20µl.

◊ In a sterile, 1.5mL microcentrifuge tube, combine the following:

  • 5.0µg high-purity total RNA
  • 2.0µl T7-oligo(dT) primer, 50µM
  • RNase-free water (variable)

◊ Incubate at 70°C for 10 minutes. 

◊ Quickly spin and put on ice.

◊ Add the following components:

  • 4µl 5X First strand cDNA buffer
  • 2µl 0.1M DTT
  • 1µl 10mM dNTP mix

◊ Mix well and incubate at 42°C for 2 minutes.

◊ Add 1.0µl SuperScript II RT (200U/µL).

◊ Mix well and gently spin down in centrifuge.

◊ Incubate at 42°C for 1 hour.

◊ Place First Strand reactions on ice for 2 minutes and centrifuge briefly to bring down condensation on sides of the tube.

◊ To the First Strand synthesis tube, add the following:

  • 91µl DEPC-treated water
  • 30µl 5X Second Strand Reaction Buffer
  • 3µl 10mM dNTP mix
  • 1µl E. coli DNA ligase (10U/µl)
  • 4µl E. coli DNA Polymerase I (10U/µl)
  • 1µl E. coli RNase H

        Final volume is 150µl.

◊ Gently tap the tube to mix.

◊ Briefly spin down to remove condensation.

◊ Incubate at 16°C for 2 hours in a cooling water bath.

◊ Add 2µl T4 DNA Polymerase (10U).

◊ Return to 16°C water bath for 5 minutes.

◊ Add 10µl 0.5M EDTA.

◊ Proceed with cDNA clean up or store crude cDNA overnight at -20°C.


Cleanup of Double-Stranded cDNA

◊ Using the GeneChip Sample Cleanup Module, add 600µl cDNA Binding Buffer to the sample.

◊ Mix for 3 seconds and check that the color of the mixture is yellow.

◊ Add 500µl of the sample to a cDNA Cleanup Spin Column sitting in a 2mL Collection Tube and centrifuge for 1 minute at 10,000rpm.

◊ Discard flow-through.

◊ Reload the column with the remaining sample (~262uL) and centrifuge as above.

◊ Discard flow-through and Collection Tube.

◊ Transfer spin column to a new 2mL Collection Tube.

◊ Pipet 750µl cDNA Wash Buffer onto spin column and centrifuge for 1 minute at 10,000rpm.

◊ Discard flow-through.

◊ Open the cap of the spin column and centrifuge for 5 minutes at max. speed.

◊ Discard flow-through and Collection Tube.

◊ Transfer spin column into a 1.5mL Collection Tube.

◊ Pipet 14µl of cDNA Elution Buffer directly onto spin column membrane.

◊ Incubate for 1 minute at room temperature.

◊ Centrifuge for 1 minute at max. speed to elute. (average volume of eluate is about 12µl).

◊ Proceed with IVT labeling.


Synthesis of Biotin-Labeled cRNA

◊ Thaw components of the ENZO BioArray RNA Transcript Labeling Kit and purified cDNA mixture from the previous day.

◊ In a new, sterile, 1.5mL RNase-free microfuge tube, add the following components:

  • 10µl purified cDNA
  • 12µl RNase-free water
  • 4µl 10X HY Reaction Buffer (vial#1)
  • 4µl 10X Biotin-Labeled Ribonucleotides (vial#2)
  • 4µl 10X DTT (vial#3)
  • 4µl 10X RNase Inhibitor Mix (vial#4)
  • 2µl 20X T7 RNA Polymerase (vial#5)

        Total reaction volume is 40µl

◊ Gently mix and spin down.

◊ Incubate in a 37°C water bath for 4 hours.


Cleanup of IVT Reaction

◊ Take out 1ul of the reaction mix for gel electrophoresis inspection.

◊ Using the GeneChip Sample Cleanup Module, add 60µl of RNase-free water to the reaction.

◊ Mix for 3 seconds.

◊ Add 350µl IVT cRNA Binding Buffer and mix for 3 seconds.

◊ Add 250µl of 100% ethanol and mix well (do not centrifuge).

◊ Add sample (~700ul total) to the IVT cRNA Cleanup Spin Column sitting in a 2mL Collection Tube.

◊ Centrifuge for 15 seconds at 10,000rpm.

◊ Discard flow-through and Collection Tube.

◊ Transfer spin column to a new 2mL Collection Tube.

◊ Add 500µl IVT cRNA Wash Buffer to spin column.

◊ Centrifuge for 15 seconds at 10,000rpm.

◊ Discard flow-through.

◊ Add 500µl of 80% ethanol onto spin column.

◊ Centrifuge for 15 seconds at 10,000rpm.

◊ Discard flow-through.

◊ Open the cap of the spin column and centrifuge for 5 minutes at max. speed.

◊ Discard flow-through and Collection Tube.

◊ Transfer spin column to a new 1.5mL Collection Tube and pipet 11µl of RNase-free water directly onto the spin column membrane.

◊ Centrifuge for 1 minute at max. speed to elute.

◊ Add another 10µl of RNase-free water directly onto spin column membrane and centrifuge for 1 minute at max. speed to elute.

◊ Use spectrophotometric analysis to determine cRNA yield and purity using a 1:100 dilution of purified cRNA (A260/A280 ratio should be close to 2.0 for pure RNA).

◊ Calculate the adjusted cRNA yield using the following:

                       adjusted cRNA yield = RNAm – (total RNAi)(y)

                       RNAm = amount of cRNA measured after IVT (µg)

                       total RNAi = starting amount of total RNA (µg)

                       y = fraction of cDNA reaction used in IVT

    ◊ For fragmentation, adjusted cRNA concentration must be at least 0.6µg/µl.

    ◊ The final concentration of RNA in the fragmentation mix can range from 0.5µg/µl to 2µg/µl.

    ◊ For standard arrays, 15µg of fragmented cRNA is required.

    ◊ In a thermal cycler compatible microfuge tube, combine 4µl 5X Fragmentation Buffer (supplied in GeneChip Sample Cleanup Module), 15µg cRNA (variable) and RNase-free water for a final volume of 20µl.

    ◊ Incubate in a thermal cycler at 94°C for 35 minutes.

    ◊ Place mixture on ice.

    ◊ Take out 1ul of fragmented cRNA for gel electrophoresis inspection.

    ◊ Run crude cRNA and fragmented cRNA samples, along with a 1Kb ladder on a 1% agarose gel for visual inspection.


    Hybridization

    ◊ Thaw components of the Eukaryotic Hybridization Control Kit.

    ◊ Heat 20X Eukaryotic Hybridization Control to 65°C for 5 minutes to denature cRNA controls.

    ◊ Equilibrate desired probe array to room temperature.

    ◊ In a new, sterile RNase-free 1.5mL microfuge tube, add the following components:

    • 20µl fragmented cRNA
    • 5µl Control Oligonucleotide B2 (3nM)
    • 15µl 20X Eukaryotic Hybridization Controls
    • 3µl Herring Sperm DNA (10mg/mL)
    • 3µl Acetylated BSA (50mg/mL)
    • 150µl 2X Hybridization Buffer
    • 104µl RNase-free water

            Total hybridization volume is 300µl

    ◊ Heat hybridization cocktail to 95°C for 5 minutes, followed by heating at 45°C for 5 minutes.

    ◊ In a new, sterile RNase-free 1.5mL microfuge tube, combine a volume of 2X Hybridization Buffer with an equal volume of RNase-free water (make 1X Hybridization Buffer).

    ◊ Wet the probe array by filling it with 250µl of 1X Hybridization Buffer and incubate in Hybridization Oven 640 set at 45°C for 10 minutes with rotation setting at 60rpm.

    ◊ Centrifuge hybridization cocktail for 5 minutes at max. speed to spin down any insoluble material.

    ◊ Remove 1X Hybridization Buffer from probe array and fill with 250µl of hybridization cocktail, taking care not to pipette any insoluble material.

    ◊ Place probe array in Hybridization Oven 640, heated to 45°C and with rotation setting at 60rpm for 16 hours.

     

    Day #3

    Washing, Staining, and Scanning

    ◊ For each array, in a new, sterile RNase-free 1.5mL microfuge tube, combine the following:

    • 600.0µl of 2X MES Stain Buffer
    • 48.0µl Acetylated BSA (50mg/mL)
    • 12.0µl Streptavidin Phycoerythrin (SAPE) (1mg/mL)
    • 540.0µl water, Molecular Biology Grade

            Total volume is 1200µl.

    ◊ Divide into two aliquots of 600µl each to be used for stains 1 and 3.

    ◊ For each array, in a new, sterile RNase-free 1.5mL microfuge tube, combine the following:

    • 300.0µl 2X MES Stain Buffer
    • 24.0µl Acetylated BSA (50mg/mL)
    • 6.0µl Norma Goat IgG (10mg/mL)
    • 3.6µl Biotinylated antibody (0.5mg/mL)
    • 266.4µl water, Molecular Biology Grade

            Total volume is 600µl.

    ◊ After 16 hours of hybridization, remove 250µl of sample (in hybridization cocktail) and put back into eppendorf, containing fragmented cRNA sample.

    ◊ Fill the array with 250µl of Non-Stringent Wash Buffer (Wash A)

    ◊ Now ready for Fluidics Station, followed by scanning.