Section 1: GeneChip Expression Analysis Overview
Contents
1. to ensure accuracy as incorrect measurement of reagents may affect the outcome of the procedure Instruments The Affymetrix GeneChip Expression Analysis Technical Manual is designed for use in a system consisting of a Fluidics Station a Hybridization Oven 640 and a Scanner References 1 Sambrook J Fritsch E F Maniatis T Molecular Cloning A Laboratory Manual v 1 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY p 21 52 1989 2 See www affymetrix com for current GeneChip technology references Limitations m The results of the assay kit are dependent upon the quality of the input RNA subsequent proper handling of nucleic acids and other reagents m The results should be evaluated by a qualified individual Q IMPOR Do not store enzymes in a frost free freezer AFFYMETRIX u Overview2. Section 1 GeneChip Expression Analysis Overview 701022 Rev 2 GeneChip Expression Analysis Overview Introduction and Objectives 2 en 1 1 4 Explanation of GeneChip Probe Arrays KK KI K K K K 00000004 1 1 4 GeneChip Expression Analysis Overview KK KI KI KI KI K K KI KI 00000 1 1 5 Precautions aa DD DD a a HH HH HHHH HH HH HHHH E E E E E HHHH He 1 1 6 Terminalo ye ye AA bas ee Ke Mr n aa Q E hiya ana Ss ah ee Ke ee as 1 1 7 Interfering Conditions 2 KK KK KK KK KEK KI KI KI KI KI KK KK KK KK KK 1 1 7 TMStrUMeNts 5 33 x 2 6 3 Aare eek ae alah a a a argent lawl en Bete oo 1 1 7 RELCTENCES 5 252 take te fe ca Pie WE ce Bane Ate ie ae ce ge he A HHH HE 1 1 7 LAMICTAL ONS 5 Mee hee foi tired dice a pared to r oppmnmm 1 1 7 This Chapter Contains a An overview of GeneChip Expression Analysis m A summary of the procedures covered in the remainder of the manual 701023 Rev 3 1 1 3 SECTION 1 GeneChip Expression Analysis Overview Introduction and Objectives Welcome to the Affymetrix GeneChip Expression Analysis Technical Manual This manual is a technical guide for using GeneChip expression analysis probe arrays All protocols included in this manual have been used successfully by scientists at Affymetrix or have been recommended by our collaborators during the development of particular products The field of mRNA gene expression monitoring is rapidly evolving and perio
3. al regulations This includes adherence to the OSHA Bloodborne Pathogens Standard 29 CFR 1910 1030 for blood derived and other samples governed by this act Never pipet by mouth Avoid specimen contact with skin and mucous membranes 3 Exercise standard precautions when obtaining handling and disposing of potentially carcinogenic reagents 4 Exercise care to avoid cross contamination of samples during all steps of this procedure as this may lead to erroneous results 5 Use powder free gloves whenever possible to minimize introduction of powder particles into sample or probe array cartridges CHAPTER 1 GeneChip Expression Analysis Overview Terminology Probes The oligonucleotides on the surface of the probe arrays are called probes because they probe or interrogate the sample Target The target is the labeled nucleic acid that is being interrogated It is hybridized to the probes on the array Probe Cell Specific areas on the probe array that contain oligonucleotides of a specific sequence Interfering Conditions C AUTI Wear powder free gloves throughout procedure Take steps to minimize the introduction of exogenous nucleases Water used in the protocols below is molecular biology grade nuclease free Proper storage and handling of reagents and samples is essential for robust performance All laboratory equipment used to prepare the target during this procedure should be calibrated and carefully maintained
4. dic technical updates to this manual will reflect the newest protocols and information for using GeneChip probe arrays This manual applies to all GeneChip expression products As an Affymetrix GeneChip user your feedback is welcome Please contact our technical support team with any input on how we can improve this resource Explanation of GeneChip Probe Arrays GeneChip probe arrays are manufactured using technology that combines photolithography and combinatorial chemistry Tens to hundreds of thousands of different oligonucleotide probes are synthesized on each array Each oligonucleotide is located in a specific area on the array called a probe cell Each probe cell contains millions of copies of a given oligonucleotide Probe arrays are manufactured in a series of cycles Initially a glass substrate is coated with linkers containing photolabile protecting groups Then a mask is applied that exposes selected portions of the probe array to ultraviolet light Illumination removes the photolabile protecting groups enabling selective nucleoside phosphoramidite addition only at the previously exposed sites Next a different mask is applied and the cycle of illumination and chemical coupling is performed again By repeating this cycle a specific set of oligonucleotide probes is synthesized with each probe type in a known location The completed probe arrays are packaged into cartridges During the laboratory procedure described in this
5. manual biotin labeled RNA or DNA fragments referred to as the target are hybridized to the probe array The hybridized probe array is stained with streptavidin phycoerythrin conjugate and scanned by the GeneChip Scanner 3000 or the GeneArray Scanner The amount of light emitted at 570 nm is proportional to the bound target at each location on the probe array CHAPTER 1 GeneChip Expression Analysis Overview GeneChip Expression Analysis Overview The following major steps outline GeneChip expression analysis 1 Target Preparation 2 Target Hybridization 3 Experiment and Fluidics Station Setup 4 Probe Array Washing and Staining 5 Probe Array Scan 6 Data Analysis Due to the differences in the RNA species between eukaryotic and prokaryotic organisms different target labeling protocols have been optimized Sections 2 and 3 provide detailed protocols for target preparation hybridization array washing and staining for eukaryotic and prokaryotic arrays respectively Please refer to the sections in this manual for detailed protocols appropriate for your arrays Step 1 Target Preparation This manual describes procedures for preparing biotinylated target from purified eukaryotic and prokaryotic RNA samples suitable for hybridization to GeneChip expression probe arrays These procedures are recommendations only For more information on these procedures please contact Affymetrix Technical Support at 1 888 DNA CHIP
6. or 44 0 1628 552550 in Europe For eukaryotic samples using protocols in this manual Section 2 double stranded cDNA is synthesized from total RNA or purified poly A messenger RNA isolated from tissue or cells An in vitro transcription IVT reaction is then done to produce biotin labeled cRNA from the cDNA The cRNA is fragmented before hybridization For prokaryotic samples Section 3 describes a detailed protocol to isolate total RNA followed by reverse transcription with random hexamers to produce cDNA After fragmentation by DNase I the cDNA is end labeled with biotin by terminal transferase Step 2 Target Hybridization A hybridization cocktail is prepared including the fragmented target probe array controls BSA and herring sperm DNA It is then hybridized to the probe array during a 16 hour incubation The hybridization process is described in the respective sections for the different probe array types AFFYMETRIX u Overview SECTION 1 GeneChip Expression Analysis Overview Step 3 Experiment and Fluidics Station Setup Specific experimental information is defined using Affymetrix Microarray Suite on a PC compatible workstation with a Windows 2000 operating system The probe array type sample description and comments are entered in Microarray Suite and saved with a unique experiment name The fluidics station is then prepared for use by priming with the appropriate buffers For more information on the fluidics s
7. tation refer to the Fluidics Station User s Guide Step 4 Probe Array Washing and Staining Immediately following hybridization the probe array undergoes an automated washing and staining protocol on the fluidics station Step 5 Probe Array Scan Once the probe array has been hybridized washed and stained it is scanned Each workstation running Affymetrix Microarray Suite can control one scanner The software defines the probe cells and computes an intensity for each cell Each complete probe array image is stored in a separate data file identified by the experiment name and is saved with a data image file dat extension Review the scanner user s manual for safety precautions and for more information on using the scanner Step 6 Data Analysis Precautions Data is analyzed using the Microarray Suite Expression Analysis window The dat image is analyzed for probe intensities results are reported in tabular and graphical formats Information on data analysis is provided in the enclosed GeneChip Expression Analysis Data Analysis Fundamentals booklet P N 701190 A WARN 1 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES 2 Avoid microbial contamination which may cause erroneous results All biological specimens and materials with which they come into contact should be handled as if capable of transmitting infection and disposed of with proper precautions in accordance with federal state and loc
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