A54011 ADAugust 2014

© 2014 Beckman Coulter, Inc.

GeXP Gene Expression Workflow

250 S. Kraemer Blvd.Brea, CA 92821

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Multiplex Design using NCBI Primer-BLAST

Pre-Design Considerations 1. Choose a correct Accession Number for each gene target.

NOTE When possible, always use reference sequence (RefSeq) (NM_XXXXXX).

2. For genes with transcript variants, decide how many variants to detect. 3. Search for possible gene family members, pseudogenes for each gene target

Primer Design using NCBI Primer-BLAST Design primers for each gene in the multiplex following steps 1 to 7 below. Ensure the amplicons are at least 5 nt apart.

1. Connect to: http://www.ncbi.nlm.nih.gov/tools/primer-blast/ 2. In “PCR Template” box, enter accession number or FASTA sequence.3. Under “Primer Parameters”, set PCR product size range as 105 to 350 nt.

NOTE For FFPE samples, set PCR product size range as 63 to 150.

4. In “Exon/Intron selection:” r For “Exon junction span”, select “Primer must span an exon-exon

junction.”

r Check box for “Intron Inclusion” and modify Min. Intron length as needed.

5. For “Primer Pair Specificity Checking Parameters:” r Check mark the box for “Specificity check.”

r Select Refseq mRNA as Database; select appropriate organism.

6. Click on “Advanced parameters” hyperlink: r Check mark the box for “Primer binding site may not contain known SNP.”

r Change Salt correction formula to “Schildkraut and Lifson 1965.”

r Change “Table of thermodynamic parameter” to “Breslauer et al. 1986.”

7. Click on the “Get Primers” button. Assemble the multiplex in a Microsoft Excel worksheet.

Post-Design Considerations1. Perform BLAST with each primer sequence to ensure it does not have

significant homology to amplicon sequences for other genes in the multiplex.2. If a primer has significant homology to a region of an amplicon of another gene

in the multiplex, re-design the primer.3. Before placing primer orders, add the forward universal tag to the 5’ end of

forward primers. Add the reverse universal tag to the 5’ end of reverse primers.

Multiplex Build and Primer Evaluation

Make 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 (or 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, TE buffer, or nuclease-free water is recommended.

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 no forward primer or with

singlet or multiplex forward primers.

Perform Evaluation: Dilute PCR reactions in 10 mM Tris-HCl, pH 8 as needed for detection within the linear range of the instrument. Perform sample run. Evaluate results in Fragment Analysis Module.

Multiplex Optimization

Redesign Primers: Redesign a specific set(s) of primers using NCBI Primer-Blast 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, use the same KANr RNA concentration for the entire study.

NOTE Every time a stock solution is thawed, make 10 μL aliquots immediately after thawing. Store aliquots at -80°C.

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. 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. 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 Curve

Prepare 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 Protocol

Prepare 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 GeXP

Prepare PCR Samples

1. Combine 1 µL 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 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 3 times, and Gel Capillary Fill 3 times. 5. Perform an Optical Alignment. 6. Perform Monitor Baseline.

Setup, Load, and Run Sample Plate

1. Name sample wells. 2. Assign Frag-3 method and Sensitive GeXP Analysis Parameters to each well.

(Slope threshold = 1, Relative peak height threshold = 0%) 3. Save the plate setup to the appropriate project. 4. Refresh the wetting tray with DI water. 5. Load sample and buffer plates and start the run.

View Fragment Analysis Study1. Open Fragment Analysis Module and create a new study with analyzed results.2. In “Results Set View,” verify that all multiplex peaks are present in Reference

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

Perform Binning and Setup Locus Tag and Allele IDs1. Select “Analysis” and “New Binning Analysis.”

NOTE A minimum of 10 results is required for binning.

2. Default parameters for Human ReferencePlex: r Dye: D4:

r Fragment Range: 149 to 340

r Max. Bin Width: 1

r Min. Data Point per Bin: 2

r Repeat Unit Length: 1

r Allele Naming convension: Alphabetic

3. Click on “Next”, and adjust “Min Rel Peak Height:” 4. Sort “Allele List” by “Num. Points,” and then delete empty bins. 5. Screen for noise peaks, shoulders, and “- A” peaks. Delete them if present.6. Sort by nominal size (nt) and place it back to order of fragment size.7. Rename allele IDs with gene names (≤ 8 characters and numbers).8. Select “Locus Tag” tab and type identical name under the “Locus Tag” and

“Locus Name.”

NOTE Do not alter the Locus Information.

9. Select “Allele ID Criteria” tab and “Overwrite system confidence interval.”10. Review the source data of all included samples, then click on ‘Finish.”11. In “Results Set View”, select and right click on data files to be reanalyzed.12. Select “Reanalyze Results” and “Using Additional/Edited Locus Tags.”13. Select the Locus Tag and click on “Next.”14. Click on “Analyze”, then click on “Finish.”15. In "Fragment List" view, set up and apply an exclusion filter: allele ID = (blank or

empty space).16. Export analyzed data via “Transfer fragments for GeXP…” as a .csv file.

GeXP Data Tool

1. Download GeXP Data Tool from BCI website. 2. Launch GeXP Data Tool, select "Open," and choose Data File.

3. Perform Normalization against Kanr . Export Data as a .txt file.

GeXP Quant Tool Analysis1. Downlaod GeXP Quant Tool from BCI website.2. Open the “Standards” file, and then open the "Samples" file.3. Choose the reference gene(s) for normalization.4. Perform "Run Quantitative Analysis."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.

GeXP Gene Expression Workflow