A54011 ACDecember 2009

GenomeLab GeXP Reference Workflow

Administrator ModuleEstablish User Account(s): Assign a username and password.Import Multiplex TDF files: Import multiplex TDF files for analysis of multiplexes not designed with this eXpress Profiler unit.Enter Genes: Create a proprietary gene sequence with a unique accession number using the Manage Genes function.Enter Primers: Enter primers created with third-party software and Save as Reference Primers using the Manage Primers function.

Multiplex Design with eXpress DesignerEnter Genes: Use the FASTA function to enter gene sequences with GenBank accession numbers.Design Individual Sets of Primers: Create a forward and reverse primer to a specific a gene with increased design control using the Primer Design function. Also save primers created with third-party software to database using the Advanced Options function.

Design a Custom Multiplex Create a multiplex by entering accession numbers using the eXpress Designer function. Create a multiplex by choosing previously saved genes and/or primers using the Multiplex Designer function. For both methods: 1. Assign a unique Multiplex Name.

2. Choose Reference Primers (including KANr).

3. Adjust multiplex design settings as needed, then click Execute to create the multiplex.

4. Click Save to Database once the multiplex is completed.

Quality Control of Multiplex Primers and Amplicon Sequence using NCBI BLAST 1. Perform BLAST for each amplicon sequence to check for highly homologous

sequences in the primer region that may lead to the production of undesigned peaks (UDPs).

2. Check each amplicon sequence with BLAST-SNP to ensure that 1) there are no single nucleotide polymorphisms (SNPs) within the primer sequences and 2) there are no repeat sequences are present in the amplicon.

3. Redesign primer(s) with the Primer Design function if any of these conditions exist for a particular primer or gene product.

4. Incorporate the redesigned primer set(s) into the multiplex and remove the unwanted primers using the Multiplex Designer function.

Export TDF file and Order Primers1. Export the multiplex TDF file.2. Open the file with Microsoft Excel to view the primer sequences.3. Order the primers in the columns N and O that are labeled Left Sequence with

Universals and Right Sequence with Universals, respectively. Do not order the

KANr primers which are provided as part of the GeXP Start Kit.

Multiplex Build and Primer EvaluationMake Primer Stocks: Suspend primers to a stock concentration of 100 µM in 10 mM Tris-HCl, pH 8.Build Multiplexes: Mix primers in a total of 1 mL with 10 mM Tris-HCl, pH 8 to create a 10X Forward Multiplex (200 nM) and a 10X Reverse Multiplex (500 nM).Prepare Forward Singlet Primers: Dilute individual forward primers in a total of 1 mL with 10 mM Tris-HCl, pH 8 to create 10X Forward Singlet Primer (200 nM) suspensions for forward singlet reactions.Formulate a custom Reference RNA: Mix untreated and treated (normal and tumor) sample total RNA to create a Reference RNA with all multiplex gene transcripts represented. Prepare a working stock 100ng/uL. 50 ng total RNA per reaction is the initial recommended concentration. Increase or decrease the total RNA for multiplex reactions based on the overall signal intensity.

Preliminary KANr Concentration: Initially a 1:50 dilution of the KANr RNA with RNA storage solution (1 mM sodium citrate), TE buffer or nuclease-free water is

recommended. Dilute enough KANr RNA to use for all of the multiplex and singlet

evaluation reactions. Use this initial dilution as the preliminary KANr RNA

concentration and optimize the final concentration based on KANr peak height relative to the multiplex peaks.Prepare Singlet and Multiplex evaluation reactions: 1. Perform RT reaction with Reverse Multiplex and Reference RNA, including NTC

and RT minus controls. 2. Pool Standard RT reactions, then perform PCR with singlet or multiplex forward

primers.

Pre-dilution: Dilute one multiplex PCR reaction (1:5, 1:10, 1:20) in 10 mM Tris-HCl, pH 8 to determine best dilution for detection within the linear range of the instrument.

Multiplex OptimizationRedesign Primers: Redesign a specific set(s) of primers using the Primer Design function to remove any significant UDPs or co-migrating peaks from the multiplex.

Perform KANr Optimization: Adjust the concentration of KANr RNA per reaction such

that the signal strength of the KANr peak is just below the median range of the

multiplex. The KANr optimization can be performed simultaneously with Attenuation.

Once the optimal KANr RNA concentration is determined, the original stock solution should be diluted in bulk using RNA Storage Solution or TE and stored at -80°C in small aliquots.Perform Attenuation: Bring the signal strength of high expressers down by adjusting the reverse primer concentration for each high signal peak. 1. Prepare two 500 nM pools of primers:

r those to be Attenuated r those to remain at the standard concentration

(Non-attenuated). 2. Prepare two-fold dilutions of the Attenuated primer pool over 8 dilutions in 10

mM Tris-HCl, pH 8. 3. Combine 2 µL of the Non-attenuated pool with 2 µL of each Attenuated pool

dilution in separate RT reactions. It is recommended that primer attenuation reactions be evaluated in duplicate (16 reactions total).

4. Perform the PCR reactions with the optimized forward multiplex.

© 2009 Beckman Coulter, Inc.

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5. Evaluate the products on the GeXP System to determine the best concentration for each reverse primer.

6. Run duplicate reactions on inner and outer capillaries. 7. Repeat the RT-PCR reactions with the fully attenuated reverse multiplex and

optimized KANr RNA concentration to verify the optimization.

8. Re-attenuate and/or modify KANr concentration as necessary to create a balanced profile best suited for detecting changes in gene expression.

9. The optimized multiplex is ready to be used to create a Standard Curve and evaluate Experimental samples with the Standard GeXP Chemistry Protocol.

Create a Standard CurvePrepare Reference RNA dilutions: Create a series of ten two-fold dilutions of the Reference RNA ranging from 100ng/uL down to 0.2ng/µL.Prepare Standard Curve RT-PCR Reactions: Prepare at least three replicate RT and PCR reactions for each Reference RNA dilution per the Standard GeXP Chemistry

Protocol. Use the optimized KANr RNA concentration in each RT reaction.

NOTE A high quality Standard Curve only needs to be run once for each optimized multiplex on a GeXP system, as long as the experimental samples fall within the standard curve.

Standard GeXP Chemistry ProtocolPrepare RT Reaction: Perform multiplex reactions with optimized reverse multiplex primers, the optimized KANr RNA concentration and Reference RNA or Experimental sample RNA (5 - 100 ng total RNA). Include NTC and RT minus control reactions. Prepare PCR Reaction: Perform amplification with optimized forward multiplex primers and RT reaction. Pre-dilution: Dilute PCR reactions in 10 mM Tris-HCl, pH 8, as needed, to bring signal into linear range of detection.

GenomeLab GeXPPrepare PCR Samples 1. Combine 1µL of the PCR reaction (or pre-dilution) into 38.5 µL SLS + 0.5 µL ss400,

mix and cover with 1 drop mineral oil. 2. Prepare the buffer plate by placing 250 µL of Separation Buffer into each well.

Setup GeXP 1. Create a new project and rename the project in the appropriate experimental

database.2. Install capillary array and gel cartridge. 3. Preheat capillaries to 50°C. 4. Perform Manifold Purge with 0.4 mL gel, at least three times. 5. Perform Gel Capillary Fill 3 times. 6. Perform an Optical Alignment. 7. Perform Monitor Baseline.

Setup Sample Plate1. Name sample wells. 2. Assign Frag-3 method and Sensitive GeXP Analysis Parameters (Slope

threshold = 1%, Relative peak height threshold = 0) to each well. 3. Add notes to each well in Notes window, if desired. 4. Export the data from all the wells to a folder on the desktop, if desired.5. Save the plate setup to the appropriate project.

Load and Run Sample Plate1. Refresh the wetting tray with DI water. 2. Load sample and buffer plates and start the run.

View Fragment Analysis Study1. Open Fragment Analysis program. 2. Create a new study with the analyzed results. 3. Apply the appropriate exclusion filter in the Fragments List window. 4. Verify that the current profiles are good. 5. View the Analyzed Data. 6. Verify that all multiplex peaks are present in Reference RNA samples and that

all gene peaks are called as single peaks. Exclude any overrange peaks that are split into two called peaks. Exclude any data with abnormal current, poor resolution of fragments and the NTC and RTminus controls.

7. Export Data via Transfer fragments for GeXP from Fragment Analysis program.

eXpress AnalyzerCreate GeXP Analysis1. Create a New Analysis. 2. Assign a multiplex to this analysis. 3. Import GeXP Fragment Analysis data.

NOTE Make sure to import all of the plates at once.

Perform Plate Setup Perform the following steps for each plate in the study and save after each step. 1. Perform a Sample Layout, which creates the association between the sample

well and the multiplex. 2. Perform Peak Binning which aligns designed fragment size and GeXP

estimated fragment size.

Perform GeXP Analysis Normalization1. Choose KANr as the Normalization gene from the list of genes within the

multiplex.2. Select the report format: Profile (by gene).3. Export report as a text (.txt) file for further analysis.

Quant Tool1. Open the Standards .txt file.2. Open the Experimental samples .txt file.3. Choose the reference (housekeeping) genes for normalization.4. Run the Quant Tool.5. Assess the Excel file for normalized gene expression values (GEQ Norm) taking

note of any experimental sample values that may be outside the range of the Standard Curve for a particular gene.

6. Calculate fold-changes in expression for a particular gene by dividing the GEQ Norm value of one sample by the GEQ Norm value of another sample.

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GenomeLab GeXP Workflow