GeXP Chemistry Protocol

Transcription

1 GeXP Chemisty Potocol GenomeLab Genetic Analysis System A29143AD Decembe 2009 Beckman Coulte, Inc. 250 S. Kaeme Blvd. Bea, CA 92821

2 GeXP Chemisty Potocol GenomeLab Genetic Analysis Systems A29143AD (Decembe 2009) Copyight 2009 Beckman Coulte, Inc. All ights eseved. No pat of this document may be epoduced o tansmitted in any fom o by any means, electonic, mechanical, photocopying, ecoding, o othewise, without pio witten pemission fom Beckman Coulte, Inc. Tademaks Following is a list of Beckman Coulte tademaks GenomeLab All othe tademaks ae the popety of thei espective ownes. Find us on the Wold Wide Web at:

3 Table of Contents Section 1 Peface Multiplex Gene Expession Pepaing to Use the GeXP System Reagents and Mateials Equipment Wokspace Template-Fee Aea (Reagent Pepaation Aea) Template-Addition Aea (RNA Wok Aea) GeXP System Run Sample Pepaation Aea Selecting the RNA Template fo Mulitplex Optimization RNA Isolation Quantitation/Nomalization Section 2 Multiplex Design and Optimization Oveview Designing Multiplex Pimes Identifying Multiplex Genes Choosing Refeence Genes Designing Multiplex Pimes Evaluating Pime and Amplicon Sequences Using NCBI BLAST Seaches Testing Individual Pime Pais (Singlets) Testing the Fowad Singlet -- Revese Plex Testing the Fowad Singlet -- Revese Singlet Chemisty Potocol fo Singlet Reactions Revese Tansciptase Reaction Calculating Reagent Volumes Pepaing the No-Template Contol (NTC) Reaction Mix Pepaing the RT Minus Contol Reaction Mix Pepaing the Standad Reaction Mix Running the RT Reaction PCR Reaction Calculating Reagent Volumes Pepaing the PCR Reaction Mix Pepaing Plates fo PCR Running the PCR Reaction Testing and Optimizing Multiplex Pimes Toubleshooting Pime Design fo Multiplex Optimization High Signals Missing Signals Co-Migating Peaks Unintended Peaks Attenuating GeXP Signals Detemining the Appopiate PCR Poduct Dilution Detemining the Revese Pime Concentation fo High-Expessing Genes GenomeLab GeXP Chemisty Potocol iii

4 Analyzing Attenuation Results Optimizing the KAN RNA Concentation Confiming the PCR, Revese Pime and KAN Dilutions on the GeXP System Using the Optimized Custom Multiplex Section 3 Multiplex Gene Expession Analysis Oveview Ceating a Standad Cuve Oveview Diluting the Refeence RNA Pefoming Revese Tansciptase and PCR Reactions to Ceate a Standad Cuve Loading and Running Standad Cuve Reactions on the GeXP System Pefoming Multiplex Gene Expession Analysis Oveview Revese Tansciptase Reaction Calculating Reagent Volumes Pepaing the No-Template Contol (NTC) Reaction Mix Pepaing the RT Minus Contol Reaction Mix Pepaing the Standad Reaction Mix Pepaing the RT Reaction Sample Plates Running the RT Reaction PCR Reaction Calculating Reagent Volumes Pepaing the PCR Reaction Mix Pepaing Plates fo PCR Running the PCR Reaction Loading and Running Samples on the GeXP System Running Samples on the GeXP System Analyzing Gene Expession Sample Data Section 4 Toubleshooting GeXP Reactions, Sepaations and Analysis Appendix A Technical Bulletins Additional Technical Suppot United States: Woldwide: iv GenomeLab GeXP Chemisty Potocol

5 Peface Multiplex Gene Expession 1 1Peface 1.1 Multiplex Gene Expession The GenomeLab GeXP Genetic Analysis System utilizes a patented, highly-multiplexed PCR* appoach, to efficiently look at the expession of multiplexed gene sets with sensitivity and speed. With a multiplexing capacity of analyzing up to 30 genes pe eaction, the scalable GeXP can monito tens to hundeds of genes fo up to tens of thousands of samples. GeXP uses a combined gene-specific, univesal piming stategy that convets multiplexed PCR to a two-pime pocess using univesal pimes. As a esult, the gene atio in RNA samples is maintained duing the PCR pocess. This stategy ovecomes the vaiations in amplification efficiency of diffeent genes duing the conventional amplification pocess without compomising the detection sensitivity. Pe-fomulated, eady-to-use GenomeLab GeXP Reagent kits ae optimized fo effotless and obust, quantitative gene expession. The eagent kits ae used fo multiplexing use-defined genes. Pe-designed GenomeLab GeXP multiplex gene set kits ae available fo pefoming quantitative gene expession studies on human o at genes. * The PCR pocess is coveed by patents owned by Roche Molecula Systems, Inc. and F. Hoffman La Roche, Ltd. GenomeLab GeXP Chemisty Potocol 1

6 Peface Multiplex Gene Expession The flow chat below depicts the GeXP System wokflow. 2 GenomeLab GeXP Chemisty Potocol

7 Peface Pepaing to Use the GeXP System Pepaing to Use the GeXP System Reagents and Mateials GenomeLab GeXP Multiplex Gene Set Kit* OR Custom multiplex fowad and evese pimes GenomeLab GeXP Stat Kit (A21019), containing: RT Buffe 5X, 480 µl DNase/RNase Fee H 2 O, 1200 µl Revese Tansciptase, DNA Size Standad-400, 55 µl 120 µl at 20 units/µl PCR Buffe 5X, 480 µl Mineal Oil, 5 ml KAN RNA with RI, 600 µl Sample Loading Solution, 6 ml Themo-Stat DNA Polymease, with sepaate 25 mm MgCl 2 (A25395) The RNA Stoage Solution (Ambion AM7000) Nuclease-Fee H 2 O, Non-DEPC Teated (USB o Invitogen ) 1M Tis-HCl ph 8.0 (USB 22638) GenomeLab Sepaation Buffe (608012) GenomeLab Sepaation Capillay Aay (608087) GenomeLab Sepaation Gel, 20 ml (391438), o 10 ml (608010) fo single-plate systems such as CEQ 8000 Sample Micoplates, 96-well (609801) 8-Well Cap Stips (BioRad TCS-0803 o Coning/Costa 0556) Buffe Micoplates, 96-well (609844) 2.0 ml, 1.5 ml and 0.65 ml Micotubes Aeosol Resistant Tips fo P10, P20, P100, P200 and P1000 Regula tips fo P10, P20, P100, P200 and P1000 *Fo a list of GenomeLab gene set kits, visit GenomeLab GeXP Chemisty Potocol 3

8 Peface Pepaing to Use the GeXP System Equipment GenomeLab GeXP Genetic Analysis System (A26572) Pipettos, P10, P20, P100, P200, and P1000 Micotube Centifuge Micoplate Centifuge Themal cycle with Heated Lid fo 96-Well Plates Votex Mixe Non-Fost-Fee Feezes (-80 C and -20 C) Wokspace To pevent the contamination of geneal eagents and ensue clean evese tansciptase (RT) and PCR eactions, the following pecautions and sepaated wok aeas ae ecommended when pefoming the pocedues below. Template-Fee Aea (Reagent Pepaation Aea) Use this aea fo assembling geneal eagents into eaction mixtues in a nuclease-fee and nucleic acid-fee envionment. IMPORTANT Except fo pimes and nucleotides, do not handle RNA, cdna, DNA samples o PCR poducts in this wok aea. Template-Addition Aea (RNA Wok Aea) Use this aea fo handling RNA samples such as Refeence RNA o KAN RNA. This aea will be used fo adding RNA templates to cdna eaction mixtues and cdna templates to PCR eaction mixtues. IMPORTANT Do not bing any PCR poducts into this aea. GeXP System Run Sample Pepaation Aea This aea should be used fo pepaing samples to un on the GeXP system. Manage the final PCR poducts in this aea to pevent DNA contamination in the RNA wok aea. Selecting the RNA Template fo Mulitplex Optimization It is impotant to conside the expected expession level of each gene in any paticula tissue o RNA sample when unning the initial tests of the designed pimes. Failue to detect an appopiate PCR poduct can indicate that the pime design is not optimal o that the gene is not epesented in the RNA sample tested. Knowledge of the taget gene s expession pofile in vaious tissues is essential when intepeting these initial test esults. This infomation is available fo many genes in the NCBI UniGene database. Visit Mixtues of total RNAs fom diffeent tissues o cell lines ae commecially available. These mixtues can educe the bias pesent when using a single tissue o cell line s RNA composition. Altenatively, one can use a mixtue of total RNA fom the tissues o cell lines that will be analyzed individually with the final multiplex. 4 GenomeLab GeXP Chemisty Potocol

9 Peface Pepaing to Use the GeXP System 1 Fo example, the Refeence RNA can be a mixtue of contol and teated sample mixed at 50:50 atio. If thee is moe than one teated sample, the 50% of teated sample in the stating RNA should be a combination of equal contibutions fom all teated samples. Stat by using 50 ng total RNA pe eaction. Incease o decease the total RNA fo multiplex eactions based on the oveall signal intensity. The oveall goal is to each a median state of expession fo all of the genes epesented in the multiplex. A high quality Refeence RNA consists of a mixtue of all the expeimental samples (contol and teated, nomal and disease, etc.) that will be assayed by the optimized multiplex. The mixtue should contain all the gene tanscipts epesented in the multiplex. This mixtue aims to achieve a modeate level of gene expession fo each gene epesented in the multiplex, which esults in an optimal ange fo detection of gene expession fo each gene. This is the same RNA used to evaluate and validate pimes, as well as ceate a standad cuve. Aliquots (25-50uL) of a woking stock of the Refeence RNA should be stoed at a concentation of 100ng/ul in a buffeed RNA stoage solution, such as The RNA Stoage Solution (Ambion), at -80 C. RNA Isolation The Agencout RNAPep Total RNA Isolation Kit (Agencout, A29212) is ecommended fo total RNA isolation. Follow the instuctions included with the kit. To pevent amplification of genomic data, make sue to teat the RNA samples with DNase I befoe unning gene expession analysis. Quantitation/Nomalization The quantity of RNA can be detemined by OD260, wheeas the quality of RNA can be assessed by the value of OD260/OD280. The acceptable ange of OD260/OD280 should be 1.8 to 2.0. GenomeLab GeXP Chemisty Potocol 5

10 Peface Pepaing to Use the GeXP System 6 GenomeLab GeXP Chemisty Potocol

11 Multiplex Design and Optimization Oveview 2 2Multiplex Design and Optimization 2.1 Oveview This potocol defines the pocess by which a custom multiplex is designed and optimized fo gene expession analysis. The following pocedues ae designed fo use with the GenomeLab GeXP Stat Kit. Design pimes using the expess Designe module of expess Pofile Check pime and amplicon sequences Ode pimes Test single pime sets (singlets) on Refeence RNA templates Test the multiplex on Refeence RNA templates Analyze singlets and the multiplex eactions on the GenomeLab Genetic Analysis System Pefom signal attenuation by evese pime dilution, to balance the gene expession pofile Pefom optimization of KAN RNA concentation Run optimized multiplex eactions with inceasing amounts of Refeence RNA to ceate a Standad Cuve Run optimized multiplex eactions with sample RNA templates 2.2 Designing Multiplex Pimes The expess Designe can design an initial o fist-pass multiplex, using accession numbes of taget and efeence genes. The multiplex pime and amplicon sequences ae evaluated using BLAST analysis. Individual pimes can be e-designed with Pime Design, if necessay. Finally, assemble the multiplex with the Multiplex Designe option and expot pime sequences fo odeing. NOTE Unde some cicumstances, it may be beneficial to design individual pime sets that span an inton to minimize the concen of genomic DNA contamination. Refe to fo moe infomation. Identifying Multiplex Genes 1. Once genes of inteest ae detemined, identify thei accession numbe. These identifies can be found on the National Cente fo Biotechnology Infomation (NCBI) web site at Since the sequence epesented by the accession numbe will be used by the GeXP softwae to geneate the multiplex pimes, cetain consideations should be made when selecting accession numbes. A single gene can be epesented in the GenBank database by multiple accession numbes such as those that efe to genomic sequences, patial sequences, mutations, mrna, ESTs, altenate tanscipts, splice vaiants, o pseudogenes. GenomeLab GeXP Chemisty Potocol 7

12 Multiplex Design and Optimization Designing Multiplex Pimes Ensue the following when selecting an accession numbe: a. Coect Gene Many genes have multiple names and aliases Diffeent genes can have simila names (e.g., IL2/IL2 ecepto alpha/il2 ecepto beta/il2 ecepto gamma) Veify that the gene is fom the species of inteest b. Valid Sequence Veify that the sequence contains only the lettes A, T, G, C o N Veify that the sequence is fo mrna (cdna) Avoid genomic DNA sequences with intons Veify that the sequence is still active by eviewing the accession numbe's evision histoy using the following page: RefSeq accession numbes stating with NM_ ae ideal choices fo designing pimes because they ae fom a cuated, non-edundant database of known genes maintained by NCBI. The NM_ accession pefix denotes mrna sequences Each RefSeq accession numbe coelates to an individual identified tanscipt vaiant. A gene may have moe than one RefSeq identifies Be awae of and avoid pseudogenes by BLASTing the gene against the genome befoe pime design to avoid undesigned peaks. See BLAST tool on the NCBI website ( Be awae of tanscipt vaiants befoe pime design to avoid undesigned peaks. See Entez Gene tool on the NCBI website ( NOTE expess Designe can geneate diffeent pimes fo altenative tanscipts only if specific, non-homologous sequences have been enteed as popietay genes fo each mrna isofom though the expess Pofile Administato mode. Even if altenate tanscipts have diffeent accession numbes in GenBank, the use of these diffeent accession numbes in expess Designe will not necessaily geneate unique amplicons. To amplify altenatively-spliced tanscipts, define the mrna isofom(s) of inteest and design pimes that include o exclude specific exons o bidge unique exon-exon junctions, using the Pime Design option of expess Designe. Refe to the GenomeLab GeXP Toubleshooting Guide (A53995). Mutations o epeats can also influence esults of pime design Homologous genes, pseudogenes, o conseved domains should also be consideed when choosing a gene sequence. To aid in identifying a valid accession numbe, additional tools and infomation ae available on the NCBI web site. Examples of these include Entez Nucleotide, Entez Gene, Homologene, Unigene, Blastn, o OMIM. 2. Seveal efeence genes can be used as intenal contols fo nomalizing gene expession levels acoss samples. Include the accession numbes fo these efeence genes with the taget genes. Typically these efeence genes include beta actin, GAPDH, and cyclophilin A. Altenate o additional human efeence genes can be consideed by evaluating expeimental RNA samples with GeXP Human RefeencePlex. (A54651) 8 GenomeLab GeXP Chemisty Potocol

13 Multiplex Design and Optimization Designing Multiplex Pimes 2 Choosing Refeence Genes The ability to choose multiple efeence genes and use the calculated geometic mean of expession fo these genes to nomalize expession in each well is a poweful tool in the XP-PCR pocess. Ideally, genes chosen fo nomalization puposes should be stably expessed in all samples unde all conditions. In ode to detemine which genes ae stably expessed, one can assay the efeence genes in expeimental samples with a multiplex such as the Human Refeence Plex (A54657) o with one's own custom multiplex. To monito the stability of gene expession between samples, the peak aeas of the efeence genes should be nomalized to Kan peak height in expess Analysis (see 7.6 expess Analysis Module of the GeXP Use's Guide) and the esulting elative expession values compaed between samples. Those efeence genes that have the most stable expession between samples should be chosen as nomalization genes. The geate the numbe of nomalization genes used, the lowe the chance of any one of those genes affecting the nomalization facto. Theefoe, if thee is some fluctuation in expession of one efeence gene, the effect will be diluted by the pesence of the othe efeence genes. Designing Multiplex Pimes See Using expess Designe in chapte 7 of the GenomeLab GeXP Genetic Analysis System Use's Guide (A29142), fo detailed infomation on multiplex design. 1. Ente the accession numbes of the taget and efeence genes in Accession Numbes field of the expess Designe in the expess Pofile softwae. NOTE Some of the genes can have pime set(s) available as peset Refeence Pimes, if they have been peviously enteed by the expess Pofile Administato. 2. Click on the desied pime set(s) in the Refeence Pimes field to highlight and include these pimes in the multiplex design. KAN must be highlighted fo inclusion in the multiplex. 3. Set the multiplex paametes such as sepaation distance between fagments o fagment size ange befoe executing the multiplex design pogam. Depending on the numbe of genes in the multiplex, sepaation distance should be at least 3 nucleotides but ideally 7 nt and sequence lengths fom 100 to 350 nt. 4. Assign a unique name to the multiplex. NOTE Save the multiplex with a multiplex name, that indicates that this is a fist pass multiplex design, which is diffeent fom the final multiplex name. Multiplex files cannot be deleted fom the database, afte they have been saved. Fo example: Fist Pass Multiplex Name HumanCance_FistPass Final Multiplex Name HumanCance_Final 5. Save the multiplex design settings in the expess Designe with a unique name. By doing this, the settings can be easily ecalled, allowing fo adjustment and e-unning the multiplex design pogam. 6. Click Execute to ceate multiplex. The softwae output will include gene-specific fowad and evese pime sequences, the location of the pimes within the gene sequence, and the pime melting tempeatues fo each gene. Each gene s name, accession numbe and species is also displayed fo veification puposes. GenomeLab GeXP Chemisty Potocol 9

14 Multiplex Design and Optimization Testing Individual Pime Pais (Singlets) 7. Expot the multiplex design esults which include a list of pime sequences with and without univesal pime tags added to the 5 end. The gene-specific sequences without the univesal pime tags ae used in the next step to veify the uniqueness of each pime sequence. Evaluating Pime and Amplicon Sequences Using NCBI BLAST Seaches Use the following steps to evaluate the multiplex pime and amplicon sequences fom a peliminay multiplex design. If any pime o amplicon sequence is detemined to be unsatisfactoy, then edesign the pime(s) fo the gene taget using the Pime Design function of expess Designe. See Designing Pimes in Chapte 7 of the GenomeLab Genetic Analysis System Use's Guide (A29142). 1. Use NCBI BLAST with species-specific seaches to detemine if the amplicon fo each pime set contains homologous egions and if so, whee those egions lie. BLASTing against the RefRNA database is helpful fo homology, SNPs and epeat sequences. BLASTing against a genomic database will help detemine if any pseudogene expession may be detected. If the homologous egions ae within the evese pime sequence, edesign the pime to taget a egion of low homology, paticulaly at the 3 end of the pime. If thee is extensive homology between two genes within the same multiplex, be sue to design pimes to unique egions of each gene in ode to pevent coss amplification. 2. Examine each pime fo nucleotide polymophisms (SNP) with BLAST-SNP, especially at the 3 end, which can lead to the pefeential amplification of one allele ove anothe. If a polymophism is found, edesign the pime to a moe conseved egion. 3. Examine the amplicon sequence, geneated by each set of pimes, fo epeat sequences that can led to Taq polymease slippage and esult in stutte peaks. If 7 o moe consecutive single o di-nucleotide epeats o a seies of such epeats in close poximity to each othe ae pesent, edesign the pimes to taget an amplicon without epeats. Repeat sequences can esult in polymease slippage and stutte. Any set of pimes that has the potential to geneate an undesigned peak (UDP) within the size ange of multiplex should be edesigned to pevent the poduction of a UDP. Once the pime and amplicon sequences have been eviewed, poceed with odeing and testing the multiplex pimes. Ode the pimes with the appopiate univesal tag sequence. IMPORTANT Do not ode the KAN pimes. They ae included in the GeXP Stat Kit Chemisty. 2.3 Testing Individual Pime Pais (Singlets) Afte the gene-specific pime sequences ae detemined to be suitable multiplex pimes, ode the coesponding pimes with univesal pime tags fom an oligo manufactue, such as Integated DNA Technologies, Inc. (IDT). Ode the smallest scale fo pime synthesis. This amount is usually adequate fo pime evaluation. Standad desalted, depotected oligos ae typically of sufficient puity to use in the multiplex fomat. Once eceived, esuspend a 100 µm stock solution in Resuspension Buffe (10 mm Tis-HCl, ph 8.0). Consult with the manufactues fo specific infomation on esuspending and stoage of the oligos. NOTE Fo a list of eagents, mateials and equipment, see "Pepaing to Use the GeXP System" on page GenomeLab GeXP Chemisty Potocol

15 Multiplex Design and Optimization Testing Individual Pime Pais (Singlets) 2 Testing the Fowad Singlet -- Revese Plex IMPORTANT This pocess is essential fo evaluating the pimes and optimizing the multiplex. 1. Pepae a 10x (500 nm) evese multiplex by combining each evese pime in a total of 1 ml, with Resuspension Buffe (10 mm Tis-HCl, ph 8.0). 2. Pepae individual 10x (200 nm) woking solutions fo each fowad pime, fo a total of 1 ml in Resuspension Buffe. 3. Use the chemisty potocol fo singlet eactions on page 12, to un each fowad pime in a PCR eaction on cdna poduced by the complete evese multiplex, to assess any unintended PCR poducts poduced by pime pais acoss genes. 4. Evaluate the esults of these singlet expeiments fo the following conditions: Pesence of an appopiately-sized PCR fagment Absence of undesigned peaks that would intefee with the quantitation of designed peaks in the multiplex Sufficient signal above the backgound noise of the PCR eaction If any of the pime pais fail to meet the citeia above, conside a diffeent RNA template (ideally in a sample whee the gene is expected to be upegulated) o edesign the pimes in a diffeent egion of the sequence. Testing the Fowad Singlet -- Revese Singlet NOTE This pocess is optional and especially useful fo toubleshooting pime designs. Each gene s pime pai can be tested in an independent two-step RT-PCR eaction to veify that the pimes poduce the expected fagment and assess the numbe and sizes of any unintended PCR poducts. 1. Pepae a 10x (500 nm) woking solution of each evese pime in Resuspension Buffe. 2. Pepae a 10x (200 nm) woking solution of each fowad pime in Resuspension Buffe. 3. Use the chemisty potocol fo singlet eactions below, to un each fowad pime in a PCR eaction on cdna poduced by the complementay evese pime, to assess if the intended PCR fagment is poduced. 4. Evaluate the esults of these singlet expeiments fo the following conditions: the pesence of an appopiately-sized PCR fagment the absence of exta peaks that would intefee with the quantitation of designed peaks in the multiplex a sufficient signal above the backgound noise of the PCR eaction If any of the pime pais fail to meet the citeia above, conside a diffeent RNA template (ideally in a sample whee the gene is expected to be upegulated) o edesign the pimes in a diffeent egion of the sequence. GenomeLab GeXP Chemisty Potocol 11

16 Multiplex Design and Optimization Chemisty Potocol fo Singlet Reactions 2.4 Chemisty Potocol fo Singlet Reactions Revese Tansciptase Reaction Calculating Reagent Volumes Detemine the numbe of Revese Tansciptase (RT) eactions that need to be pefomed fo the singlet pime evaluation and incease the numbe by 10%, ounding to the next eaction, to ensue adequate volume. Complete all singlet eactions in duplicate. Pepaing the No-Template Contol (NTC) Reaction Mix 1. In the Template-Fee aea, add the following eaction components into a 1.5 ml. micocentifuge tube labeled with NTC. 2. Gently mix the components by pipetting up and down. RT Minus Reaction Mix DNase/RNase Fee H 2 O RT Buffe 5X Revese Tansciptase** Volume pe Reaction 3 μl 4 μl 1 μl **Add Revese Tansciptase last to peseve enzyme activity 3. In the Template-Addition aea, pe-dilute the KAN RNA with RI in DNase/RNase Fee H 2 O. The ecommended initial dilution is 1:50 with a ange fom 1:10 to 1:100. The final KAN concentation will be optimized in late steps. Add the pe-diluted KAN solution to the RT Minus Reaction Mix pepaed above and gently mix: Reaction Mix Pe-diluted KAN RNA with RI Volume pe Reaction 5 μl 4. Keep this mixtue on ice until eady to poceed. Pepaing the RT Minus Contol Reaction Mix 1. In the Template-Fee aea, add the following eaction components into a 1.5 ml micocentifuge tube labeled with RT Minus. 2. Gently mix the components by pipetting up and down. RT Minus Reaction Mix DNase/RNase Fee H 2 O RT Buffe 5X Volume pe Reaction 4 μl 4 μl 12 GenomeLab GeXP Chemisty Potocol

17 Multiplex Design and Optimization Chemisty Potocol fo Singlet Reactions 2 3. In the Template-Addition aea, pe-dilute the KAN RNA with RI in DNase/RNase Fee H2O. The ecommended stating dilution is 1:50 with a ange fom 1:10 to 1:100. The final KAN concentation will be optimized in late steps. Add the pe-diluted KAN solution to the RT Minus Reaction Mix pepaed above and gently mix: Reaction Mix Volume pe Reaction Volume pe Mix (3 eactions) Pe-diluted KAN RNA with RI 5 μl 15 μl 4. Keep this mixtue on ice until eady to poceed. Pepaing the Standad Reaction Mix 1. In the Template-Fee aea, add the following eaction components into a 1.5 ml micocentifuge tube labeled with Standad. 2. Gently mix the components by pipetting up and down. RT Plus Reaction Mix Volume pe Reaction Volume pe Mix (40 eactions) DNase/RNase Fee H 2 O 3 μl 120 μl RT Buffe 5X 4 μl 160 μl Revese Tansciptase** 1 μl 40 μl **Add Revese Tansciptase last to peseve enzyme activity. 3. In the Template-Addition aea, pe-dilute the KAN RNA with RI in DNase/RNase Fee H 2 O. The ecommended stating dilution is 1:50 with a ange fom 1:10 to 1:100. The final KAN concentation will be optimized in late steps. Add the pe-diluted KAN solution to the RT Minus Reaction Mix pepaed above and gently mix: RT Plus Reaction Mix Volume pe Reaction 4. Keep the mixtue on ice until eady to poceed. Pepaing the RT Reaction Sample Plates Volume pe Mix (40 eactions) Pe-diluted KAN RNA with RI 5 μl 200 μl NOTE To avoid edge effects, do not use the oute edge wells of the themal cycle. 1. Add 2 µl of the 10x evese multiplex (500 nm) OR 2 µl of an individual 10x evese singlet pime (500 nm) diectly to the appopiate well of a labeled 96-well plate. NOTE The final standad concentation fo each evese pime is 50 nm in the 20 μl RT eaction. 2. Aliquot 13 µl of the RT Reaction mixes into the appopiate wells of a labeled 96-well plate. Use the 96-well plate cooles to keep the eagents cold o place them on ice. 3. Dilute the Refeence RNA to 5-20 ng/µl with DNase/RNase Fee H 2 O. The amount of Refeence RNA pe eaction can ange fom 25 ng to 100 ng depending on the gene expession levels in the samples. GenomeLab GeXP Chemisty Potocol 13

18 Multiplex Design and Optimization Chemisty Potocol fo Singlet Reactions NOTE Fo instuctions on how to fomulate a Refeence RNA fo singlet eactions, see "Selecting the RNA Template fo Mulitplex Optimization" on page Add 5 µl of the diluted Refeence RNA (5-20 ng/µl) to each Standad and RT Minus well. 5. Fo each NTC well, add 5 µl of DNase/RNase Fee H 2 O in place of RNA template. 6. Gently mix the eaction components by pipetting up and down. IMPORTANT Make sue that ai bubbles ae not induced. 7. Tightly cove the plate with the stip caps. 8. Consolidate the liquid to the bottom of the wells by biefly centifuging (1 minute at 2,000 pm). Running the RT Reaction 1. Place the RT Reaction sample plate in the themal cycle and close the lid. 2. Run the following incubation pogam with the coect eaction volume (20 µl), and a heated lid: Tempeatue 48 C 42 C 95 C 4 C Time 1 minute 60 minutes 5 minutes hold PCR Reaction Calculating Reagent Volumes Detemine the numbe of PCR eactions that need to be pefomed. Incease the numbe of eactions by 10%, ounding up to the next eaction, to ensue enough eagent volume. Complete all singlet evaluations in duplicate. Pepaing the PCR Reaction Mix Add the following to a micocentifuge tube labeled PCRmix and gently mix. Reaction Mix Volume pe Reaction 25 mm MgCl μl PCR Buffe 5X ThemoStat DNA Polymease** Total Volume 4.0 μl 0.7 μl 8.7 μl **Add ThemoStat DNA Polymease last to peseve enzyme activity. 14 GenomeLab GeXP Chemisty Potocol

19 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes 2 Pepaing Plates fo PCR NOTE To avoid edge effects, do not use the oute edge wells of the themal cycle. 1. Aliquot 2 µl of each 10x fowad singlet pime (200 nm) to the appopiate wells of a 96-well PCR plate. 2. Aliquot 8.7 µl of the PCR eaction mix into the appopiate wells of a 96-well PCR plate. 3. Biefly centifuge the contents of the RT eactions to consolidate the solution to the bottom of the tube. 4. Tansfe 9.3 µl of the template cdna samples (completed RT eactions) fom the RT eaction plate to the coesponding wells on the PCR plate. 5. Pipet up and down to mix the eaction components thooughly. IMPORTANT Make sue that ai bubbles ae not induced. 6. Tightly cove the plate with the stip caps. 7. Consolidate the liquid to the bottom of the wells by biefly centifuging (1 minute at 2,000 pm). Running the PCR Reaction 1. Place the sample plate in the themal cycle. 2. Run the following themal cycling pogam with the coect eaction volume (20 µl) and a heated lid: Step Tempeatue Time 1 95 C 2 94 C 3 55 C C* 5 N/A 6 4 C 10 minutes 30 seconds 30 seconds 1 minute Repeat steps 2-4 fo an additional 34 cycles (total of 35 cycles). Hold *The optimal extension tempeatue can vay depending on the multiplex. Check the multiplex kit inset fo specific instuctions. Fo custom multiplexes, the ecommended stating tempeatue is 70 C. Fo instuction on Loading and Running Samples on the GeXP System, see page Testing and Optimizing Multiplex Pimes The singlet RT-PCR eactions that occu within the GeXP PCR pocess, can diffe fom those in multiplexed eactions. Signals that wee high in singlet eactions do not necessaily esult in high signals in the multiplex. Theefoe, it is impotant to chaacteize the poducts of the pime pais in a multiplexed eaction. 1. Assemble a 10x (500 nm) evese multiplex pime mix by combining the evese pimes at 500 nanomola each, in Resuspension Buffe (10 mm Tis-HCl ph 8.0). GenomeLab GeXP Chemisty Potocol 15

20 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes 2. Assemble a 10x (200 nm) fowad multiplex pime mix by combining the fowad pimes at 200 nanomola each, in Resuspension Buffe. 3. Assemble the multiplex RT and PCR eactions as descibed in the geneal pocedue "Pefoming Multiplex Gene Expession Analysis" on page 24, and un them on the GeXP system. 4. Detemine if all the pime sets in the multiplex poduce a detectable signal. 5. Compae each gene's estimated fagment size in the singlet esults to the coesponding peak in the multiplex esults. Toubleshooting Pime Design fo Multiplex Optimization A multitude of factos affect the efficiency of PCR pimes in a multiplexed fomat. Unintended esults can be minimized by following the guidelines that wee peviously suggested. See "Identifying Multiplex Genes" on page 7 and "Evaluating Pime and Amplicon Sequences Using NCBI BLAST Seaches" on page 10. Additional toubleshooting could be equied to impove the quality of the multiplex assay. High Signals Any gene signals that ae close to o above the linea detection limit of the GeXP system detecto (130,000 RFU aw data, 120,000 RFU analyzed data) in the multiplex eaction will equie pe-dilution and/o pime attenuation. Refe to page 17 fo moe infomation. Missing Signals A lack of signal can be caused by intefeence fom anothe pime in the multiplex. To test fo pime intefeence, pefom duplex eactions by combining the affected pime set with each pime set of the multiplex to detemine which pime set is causing the intefeence. Altenatively, the gene could be suppessed o not expessed in the RNA sample that was used to evaluate the multiplex, test othe RNA samples to veify a gene poduct. Some genes can be undetectable unless induced in a biological system such as an animal model o cell line. Choose RNA in which the gene in question has highe epesentation. A single gene peak of the appopiate size detected in a singlet eaction indicates that a pime pai is capable of amplifying a specific gene taget. Howeve, if the signal of the gene peak is significantly lowe than othe genes in the multiplex pofile and it is not a low expesse, something may be intefeing with detection of this gene in multiplex fomat. The attenuation of high expesses can sometimes emedy this poblem. Attenuation attempts to balance the signals fom high expesses and low expesses within the linea ange of detection fo the GeXP instument. The attenuation pocess pefomed duing multiplex optimization is descibed in "Detemining the Revese Pime Concentation fo High-Expessing Genes" on page 18. Co-Migating Peaks If two o moe diffeent PCR poducts of simila size ae indistinguishable in capillay electophoesis, it is because they have co-migated and the esult is an additive effect on the peak aea. This will affect the elative quantitation accuacy fo the gene assigned to the paticula fagment size. 16 GenomeLab GeXP Chemisty Potocol

21 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes 2 Results fom the singlet expeiments above should alet the designe to any potential fo co-migating peaks. Anothe method to confim the souce of an undesigned co-migating peak is to un a Plex-1 (plex minus one) expeiment in which the multiplex is un without the pime pai suspected of contibuting the co-migating peak. If the co-migating peak is still pesent in the absence of the suspect pime pai, eview the singlet expeiments o un a vaiety of follow-up expeiments to identify the pime pai esponsible fo the undesigned poduct, then edesign the pime(s). Often, the most diect esolution is to edesign the pime(s) fo the designed peak so that this peak no longe co-migates with the undesigned peak. Unintended Peaks The unintended amplification of sequences, othe than the selected taget gene, can occu due to high homology and conseved sequences among vaious genes, undiscoveed gene sequences, pseudogenes o altenate tanscipts. Since peak identification is based on the taget gene's fagment size and elative quantitation of gene expession is based on peak aea, the elimination of the unintended signals is integal to the pocess at seveal stages. Fist, exclusion filtes can be applied to the list of identified fagments in Fagment Analysis on the GeXP system. Second, the binning of the multiplex fagments in the Peak Binning section of expess Analysis softwae excludes any signals outside of the use-adjustable bins. Thid, the expess Analysis softwae only consides the tallest peak within each bin when quantifying peak aea fo elative quantitation. To disciminate between genes with highly conseved egions and/o high homology to othe membes within the same gene family, obtain an alignment of the highly homologous genes including the gene of inteest. Seveal online tools ae available fo geneating alignments such as ClustalW[EBI, UK]: By designing pimes with one o two 3' mismatched bases to the unwanted sequence, discimination between simila sequences impoves. Many highly homologous sequences can contain dissimila 5' o 3' untanslated egions. Pime design can be diected to these egions in GeXP Pime Design section of expess Designe by excluding the appopiate egions. If an unintended peak co-migates within 3 nucleotides of a designed peak in the multiplex, has high signal and/o affects the quantitation of anothe gene peak, then edesign pimes that cause this unintended peak. Assess the significance of an unintended peak in the singlet eaction with the pimes that cause the unintended peak, when the peak height of the designed peak is below 120,000 RFU. Attenuating GeXP Signals To keep the oveall fluoescent signal within the linea ange of detection, pe-dilute the PCR poducts befoe analysis on the GeXP. To ceate balance with the est of the multiplex pofile, attenuate the signal of high expesses and KAN, as necessay. GenomeLab GeXP Chemisty Potocol 17

22 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes Detemining the Appopiate PCR Poduct Dilution 1. Run the standad GeXP multiplex potocol using 50 nm of each of the gene-specific evese pimes in each eaction. 2. Geneate the following dilutions of the esulting PCR poducts: PCR Pe-Dilution SLS/SizeStd 400 Dilution Total Dilution Undiluted Reaction 1 μl into 39 μl SLS/SS (1:40) 1:40 2 μl into 8 μl 1 μl into 39 μl SLS/SS (1:40) 1: mm Tis-HCl ph 8.0* (1:5) 2 μl into 18 μl 1 μl into 39 μl SLS/SS (1:40) 1: mm Tis-HCl ph 8.0* (1:10) 2 μl into 38 μl 10 mm Tis-HCl ph 8.0* (1:40) 1 μl into 39 μl SLS/SS (1:40) 1:800 * Pepae 10 mm Tis-HCl ph 8.0 fom 1M Tis-HCl ph 8.0 (USB 22638): Nuclease-Fee H 2 0 (USB 71786)=1:99 (v/v). 3. Detemine the optimal PCR dilution at which the: lowest gene signal is still detectable backgound noise o baseline is minimized highest gene signal has sufficient potential dynamic ange IMPORTANT If none of the dilutions allow fo sufficient dynamic ange, appopiate evese pime attenuation must be pefomed. Continue by following the attenuation pocedue to satisfy equiements fo the lowest gene signal and backgound noise as efeenced above, then evisit the dilution facto paamete once the pope attenuation has been detemined. Typically, as signals fom high expesses ae attenuated down, the weake signals incease. Although the dilution facto can bing the weak signals close to the baseline, the isk of losing the signal below the minimum detectable level is low. Detemining the Revese Pime Concentation fo High-Expessing Genes If one o seveal genes signals ae close to o above the linea ange of the GeXP system detecto (>130,000 RFU in aw data o >120, 000 RFU in analyzed data), when un at the dilution facto detemined above, they will equie attenuation by loweing the evese pime concentation elative to the othe evese pimes in the multiplex. Choose a taget gene peak fom the multiplex pofile that epesents the median peak height of the multiplex. The taget peak seves as a benchmak by which a balanced multiplex pofile is designed though optimization of evese pime concentations. The evese pimes of all peaks with signals geate than the taget peak should be attenuated. Gene peaks with signals significantly less than the taget peak should be consideed fo inceased evese pime concentation. 18 GenomeLab GeXP Chemisty Potocol

23 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes 2 Run seial dilutions of the evese pimes of the genes that will be attenuated with the emaining evese pimes at the nomal 50 nm pe RT eaction as follows: 1. Geneate a 500 nm evese pime mix without the pimes of the attenuated genes (10xRevPlex50nM). NOTE To incease the signal intensity of one o moe exceptionally low peaks, inceased amounts of evese pime (up to 1500nM) may be added to this mix fo those gene peaks. 2. Geneate a 500 nm evese pime mix of the all of the pimes that ae to be attenuated (10xRevPlexDil). 3. Seially dilute the 10x RevPlexDil pime mix with two-fold dilutions ove 8 concentations (10x concentations of 500 nm to 3.9 nm). 4. Assemble and un the RT eaction as follows (fo each eaction): Reaction Mix DNase/RNase Fee H 2 O RT Buffe Mix 5X 10xRevPlex50nM Pime Mix 10xRevPlexDil Pime Mix (dilution) Revese Tansciptase Pe-diluted KAN RNA with RI * Contol Template Total Volume 1 μl 4 μl 2 μl 2 μl 1 μl 5 μl 5 μl 20 μl * Detemine optimal KAN RNA concentation using the potocol descibed in "Optimizing the KAN RNA Concentation" on page 20 and then pe-dilute KAN RNA in a buffeed solution to stabilize RNA (e.g. Ambion's RNA Stoage Solution). 5. Pefom the standad fowad multiplex PCR potocol with cdna geneated fom the RT eaction above. 6. Run the PCR poducts on the GeXP system using the PCR dilution detemined above. Refe to the GenomeLab Genetic Analysis System Use s Guide (A29142), Chaptes 2, 3 and 6, fo detailed instuctions on unning samples on the GeXP System. Analyzing Attenuation Results Obseve the signal levels of all the multiplex poducts at each evese pime concentation. Evaluate the esults to detemine if the: Signals of the attenuated genes fall within the linea ange of the GeXP system detecto and have sufficient dynamic ange to measue changes in gene expession The ecommended ange is appoximately 2,000 to 50,000 RFU fo most genes ove all eight capillaies Signal levels of othe genes in the multiplex emain in the linea ange and have sufficient dynamic ange GenomeLab GeXP Chemisty Potocol 19

24 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes In many cases, optimal attenuation occus at diffeent pime concentations fo diffeent genes. Fo each gene, choose the concentation at which the gene peak is appoximately equal in signal to the taget gene peak. Also, attenuation of one gene signal can aise a non-attenuated gene signal to a level above the linea ange. A systematic, epetitive appoach to attenuation and pime dilution optimization is often necessay to bing all signals to the desied levels. Moe often than not, the attenuation of high expesses will esult in an inceased signal of the low expesses. Howeve, if the attenuation does not esult in an adequate incease of the low expesse signal, the evese pime concentation fo those genes can be aise up to 150 nm pe eaction (1.5 µm in evese multiplex). Once optimum pime concentations ae detemined, epeat the PCR Poduct Dilution pocedue on page 14 to detemine the appopiate PCR dilution. Optimizing the KAN RNA Concentation The pupose of testing multiple concentations of KAN RNA in the mutltiplex eaction is to optimize the KAN peak height elative to the gene peaks such that it can be used fo calculating the elative signals fo the othe genes in the multiplex. The ideal peak height fo the KAN peak is appoximately equal to o slightly below the median signal ange of the multiplex. Fist, make an initial 1:50 dilution of the KAN RNA in wate to assay with the initial multiplex evaluation eactions in ode to detemine if the amount of KAN RNA should be titated up o down. Dilute just enough KAN RNA to pefom these initial, evaluation eactions and use 5µL of the 1:50 dilution pe eaction. Based on the initial esults with the 1:50 dilution, titate up o down the amount of KAN RNA used in subsequent eactions. Fo example, if the KAN peak is too low elative to the gene peaks, use moe KAN RNA pe eaction. If the KAN peak is too high, use less. NOTE The KAN RNA concentation can be optimized togethe with evese pime concentation optimization. The optimized KAN peak height should be about half the peak height of the taget peak designated fo pime concentation optimization. 1. Dilute KAN RNA ove a seies of dilutions, eithe lowe o highe than the initial 1:50 dilution. See Table GenomeLab GeXP Chemisty Potocol

25 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes 2 Table 2.1 KAN RNA dilution seies Dilution Oiginal vial* Volume fom pevious dilution (μl) - 4* Volume of diluent # (μl) Final Concentation (femtomola) Mixed volume tansfeed to the next dilution (μl) * Final Volume (μl) Final Dilution facto undiluted 1:12.5 1:25 1:50 1:100 1:200 1:400 * Oiginal GeXP Kan RNA with RI (BCI A21041) = 254 femtomola # Diluent = The RNA Stoage Solution (Ambion) o DNase/RNase-Fee H 2 O 2. Add 5µL of each dilution of KAN RNA to a sepaate RT eaction that contains the pe-detemined optimal amount of Refeence RNA (5-100ng) pe eaction. This is the same amount of sample RNA that will be used fo expeimental multiplex eactions. NOTE It is ecommended that each KAN RNA dilution be assayed in duplicate o tiplicate. Once the optimal KAN RNA dilution is detemined fo a paticula multiplex, dilute the concentated KAN RNA in an RNA stabilization buffe such as The RNA Stoage Solution (Ambion), to ceate a stock of Optimized KAN RNA. Aliquot the stock of Optimized KAN RNA to avoid multiple feeze/thaw cycles and stoe at -80 C. Use this optimized stock concentation fo all futue eactions with the optimized multiplex. Confiming the PCR, Revese Pime and KAN Dilutions on the GeXP System Once the appopiate concentations of evese pimes and KAN RNA have been detemined fo a given Refeence RNA sample, assemble and un the optimized multiplex against a panel of RNA samples fom vaious souces. This will veify the optimized multiplex conditions fo RNA samples that can have vaying gene expession pofiles. Additional total RNA templates fom vaious species, tissues, and cell lines ae available fom a numbe of supplies. Expected expession levels, cell and tissue types, expeimental conditions, and quality of the RNA (no contaminating genomic DNA) should be consideed when assembling the panel of RNA samples. Even with diffeent expession levels between RNA samples, all of the multiplex signals should emain within the linea ange of detection. If one o moe gene signals exceeds the linea ange of detection, the high gene signal(s) must be futhe attenuated and etested against the panel of RNA samples. Adjust the amount of total RNA and/o epeat the PCR Poduct Dilution pocedue on page 14, as necessay to ensue that all peaks ae within ange. IMPORTANT Once the paametes ae optimized fo a paticula multiplex, they should emain fixed thoughout a study. GenomeLab GeXP Chemisty Potocol 21

26 Multiplex Design and Optimization Testing and Optimizing Multiplex Pimes Using the Optimized Custom Multiplex Once the eaction conditions fo a custom multiplex ae optimized, a standad cuve is established with the Refeence RNA and expeimental samples ae assayed using the standad multiplex gene expession analysis potocol. Refe to "Ceating a Standad Cuve" on page 23 o "Pefoming Multiplex Gene Expession Analysis" on page 24, fo moe infomation. 22 GenomeLab GeXP Chemisty Potocol

27 Multiplex Gene Expession Analysis Oveview 3 3Multiplex Gene Expession Analysis 3.1 Oveview The following potocol is designed fo pefoming multiplex gene expession analysis with the GenomeLab GeXP Stat Kit, in combination with a GeXP multiplex gene set kit o an optimized custom multiplex. Beckman Coulte offes seveal unique GenomeLab GeXP gene set kits. See fo moe infomation on the gene set kits. 3.2 Ceating a Standad Cuve Oveview The pupose of ceating a high-quality standad cuve with the Refeence RNA fo an optimized multiplex is to establish an equation fo each gene fom which accuate gene expession values fo expeimental (unknown) samples can be deived. The standad cuve is ceated with inceasing concentations of Refeence RNA, which tanslates to inceasing concentations of each gene. The ecommended ange of Refeence RNA fo the standad cuve is 10 data points fom 1 ng to 500 ng. While this standad cuve may be expanded in eithe diection, if necessay, fo a multiplex with paticulaly high o low expessed genes, it should only need to be ceated one-time-pe-multiplex on each GeXP system. The stoed Standad Cuve data will be used fo all subsequent multiplex gene expession analyses pefomed with a paticula multiplex. Ceate a Standad Cuve fo multiplex gene expession analysis by completing the following pocedues: Diluting the Refeence RNA in seies of two-fold dilutions Set up the RT eactions with diluted Refeence RNA Running the RT Reaction Set up the PCR eaction Running the PCR Reaction Pepae samples fo GeXP analysis Load, un, and analyze samples on the GenomeLab GeXP Nomalize data to KAN using expess Pofile softwae Expot a Standads file fo use in Quant Tool Diluting the Refeence RNA Fom the woking stock of Refeence RNA (100ng/uL) make a seies of two-fold dilutions in eithe RNA Stoage Solution (Ambion) o DNase/RNase-fee dh 2 O. See Table 3.1. It is ecommended that enough of each dilution is pepaed such that the standad cuve is pefomed in tiplicate eactions (at least thee eactions pe dilution). 5µL of each dilution of the Refeence RNA will be need pe eaction. GenomeLab GeXP Chemisty Potocol 23