GenomeLab GeXP. Troubleshooting Guide. A53995AC December 2009

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1 A53995AC Decembe 2009 GenomeLab GeXP Toubleshooting Guide Beckman Coulte, Inc. 250 S. Kaeme Blvd., Bea, CA Copyight 2009 Beckman Coulte, Inc.

2 Copyight, Licenses and Tademaks Copyight Beckman Coulte, Inc., All ights eseved. No pat of this publication may be epoduced, tanscibed, tansmitted, o tanslated into any language in any fom by any means without the witten pemission of Beckman Coulte, Inc. Licenses and Tademaks Beckman Coulte is a egisteed tademak of Beckman Coulte, Inc. GenomeLab is a tademak of Beckman Coulte, Inc. All othe tademaks and egisteed tademaks ae popety of thei espective ownes. ii

3 Table of Contents Section 1 Intoduction Section 2 Geneal Diagnostic Guidelines Evaluating the Raw Data Examining Signal Stength and Multiplex Pofile Checking the Raw Data Evaluating the Cuent Pofiles Confiming the Sepaation Cuent Causes of Cuent Abnomalities Section 3 Instumentation & Chemisty Pefoming Instument Diagnostics Running Size Standad Intepeting the Results Running the Sequencing Test Sample Intepeting the Results Testing the Chemisty Testing the GeXP Stat Kit and Expeimental Pocess Intepeting the Results Examining the Expeimental Pocess Testing Thid-Paty Poducts Intepeting the Results Testing the Expeimental RNA and Custom Multiplex Pime Intepeting the Results Section 4 Refeence Tables Multiplex Pime Design GeXP Chemisty GeXP Sepaation and Fagment Analysis expess Pofile and Analysis Section 5 Questions & Answes expess Pofile Multiplex Pime Design GeXP Chemisty and Fagment Analysis expess Analysis Section 6 Appendix A Toubleshooting How Tos 37 Toubleshooting Multiplex Design with Vebose Mode Repaiing a Coupt TDF File Ceating a Multiplex without an Intenet Connection Designing Pimes to Detect Altenative Tanscipts Modifying a Peviously Designed Multiplex Toubleshooting Guide iii

4 Impoting Two o Moe Plates into expess Analyze Section 7 Appendix B Refeence Figues 41 iv GenomeLab GeXP

5 Intoduction Oveview 1Intoduction Oveview The GeXP Toubleshooting guide has been designed to help you identify and coect the issues most commonly encounteed with the GenomeLab GeXP Genetic Analysis System. Read though the entie GeXP Toubleshooting Guide fist, befoe pefoming toubleshooting expeiments. Begin with Geneal Diagnostics, which will infom you on the data you will need and whee it can be found. Next, Instumentation & Chemisty will povide a systematic appoach to enable you to effectively diagnose the poblem. See Figue 1.1 on page 2 fo the GeXP Toubleshooting Wokflow. This seves as a visual aide fo the toubleshooting pocess. Finally, the Refeence Tables and Questions & Answes sections ae paticulaly helpful fo esolving specific poblems. These sections can also aid in apid identification of the most likely suspects fo poo esults. Toubleshooting Guide 1

6 GeXP Toubleshooting Wokflow View the data Figue 1.1 GeXP Toubleshooting Wokflow Is the poblem with the GeXP instument? Yes I m not sue whee the poblem is. Is it the poblem with the chemisty? Yes Run the Size Standad-400 Use the GeXP Stat Kit and contol multiplex gene set kit to pefom GeXP eactions Ae the peak esolution and cuent okay? No Ae thee cuent abnomalities? Yes No Do you have a KAN peak? Yes Yes No I don t have a KAN peak. I have KAN with all multiplex peaks. Peak esolution and cuent ae okay. I have poo peak esolution. Review GeXP potocol Test thidpaty poducts Test a new kit Kit and pocess ae okay. Run the Sequencing Test Sample (PN ) Yes Is the cuent okay? Do you have a KAN peak? No No I still don t have a KAN peak. I have cuent abnomalities. Go to Chemisty Toubleshooting Contact you Beckman Coulte Field Sevice Repesentative Contact you Beckman Coulte Technical Suppot Repesentative Test expeimental RNA and pimes

7 Geneal Diagnostic Guidelines Oveview 1Geneal Diagnostic Guidelines Oveview The GenomeLab GeXP povides softwae functions that will help you to identify and esolve GeXP poblems. Two key featues in this egad ae the Raw Data and Cuent Pofile. NOTE The Analyzed Data can also be used fo toubleshooting, but only afte the Raw Data and Cuent Pofile have been shown to be acceptable. Use the aw data, cuent pofile and analyzed data to detemine whethe the issue is caused by GeXP instumentation o chemisty o possibly both. Be sue to note the following aspects of each type of data fo all of the affected samples and appopiate contols: Raw and Analyzed Data Baseline fo D4 (blue) and D1 (ed) Signal stength Signal pofile (level o dop-off) Landmaks unincopoated pimes (aw only) multiplex peaks KAN peak size standad peaks See "Evaluating the Raw Data" on page 4 fo moe infomation. Cuent Pofile Ramping pofile Maximum sepaation cuent (µa) Level of cuent thoughout sepaation See "Evaluating the Cuent Pofiles" on page 6 fo moe infomation. Toubleshooting Guide 3

8 Geneal Diagnostic Guidelines Evaluating the Raw Data 1.1 Evaluating the Raw Data The aw data geneated by the GeXP is displayed in the Data Monito window of the Run Contol module, duing the actual sepaation. The data is also accessible by using Fagment Analysis. Examining Signal Stength and Multiplex Pofile Refe to the signal stength and multiplex pofile as the fist step in diagnosing poblems, when looking at aw data. Although peak heights vay fo diffeent multiplex fagments, the signal stength of D4-dye (blue) labeled multiplex peaks should be faily even acoss the sepaation afte the initial unincopoated pime dye font One exception is the positive contol, 325 nucleotide kanamycin esistance (KAN ) peak, which is usually ove-ange in most GeXP eactions All of the D1-dye (ed) labeled size standad peaks should be appopiately spaced and appoximately the same height Figue 1.1 GeXP Raw Data 4 GenomeLab GeXP

9 Geneal Diagnostic Guidelines Evaluating the Raw Data Checking the Raw Data The electopheogam shown in Figue 1.1 shows a typical GeXP sepaation with a stat time at appoximately 11 minutes fo the Fag-3 sepaation method. The signal stength at the beginning of the sepaation is ove-ange due to excess pimes emaining in the PCR eaction. This is common fo GeXP eactions. The multiplex fagments begin appeaing at appoximately 17.5 minutes fo this paticula sample. The positive contol KAN fagment appeas at appoximately 26.5 minutes. The D1-dye (ed) labeled Size Standad-400 peaks ae appopiately spaced and even in height. The data is displayed as signal intensity, measued in elative fluoescence units (RFU) vs. time. The above example shows excellent signal stength. Howeve, it is not necessay fo each sample to have such high signal in ode to obtain accuate peak aea measuements. Moe impotant is that all the peaks fall within the limits of detection (below 130,000 RFU in the aw data) and the electopheogam shows a balanced pofile with no signal dop-off. Signal Dop-Off The example of analyzed data in Figue 1.2 shows a balanced pofile, wheeas the analyzed data in Figue 1.3 shows signal dop-off. Signal dop-off is chaacteized by elatively high signal fo shote fagments and significantly lowe signal fo the longe fagments. The esult is a multiplex pofile with a downwad slope towad the lage fagments, while the size standad peaks ae even and appea nomal. The KAN peak may o may not appea to be affected by signal dop-off. Iegulaities in PCR cycling conditions, paticulaly the extension tempeatue, ae the most likely cause of signal dop-off. This is often seen in the edge wells of a themal cycle. Ensue that the themal cycle is calibated and conside using only the non-edge wells to avoid signal dop-off. See "GeXP Chemisty" on page 20 fo moe infomation. NOTE Signal dop-off can be caused by using the wong DNA Polymease. ThemoStat DNA Polymease (A25395) has been validated fo use with the GenomeLab GeXP system. Figue 1.2 GeXP Analyzed Data: Nomal Peak Pofile Toubleshooting Guide 5

10 Geneal Diagnostic Guidelines Evaluating the Cuent Pofiles Figue 1.3 GeXP Analyzed Data: Peak Pofile with Signal Dop-off IMPORTANT Signal dop-off can lead to high %CV in analyzed data. Do not use the esults of these wells fo data analysis. 1.2 Evaluating the Cuent Pofiles The GeXP instument continuously monitos the cuent in each of the eight capillaies, while the system is unning. The cuent pofile can be extemely useful in diagnosing cetain issues. The cuent pofile is displayed in the Data Monito window of the Run Module, duing the actual sepaation, and is also accessible by using Fagment Analysis. Confiming the Sepaation Cuent The figues below display a compaison of aw data fom the same sample sepaated with nomal cuent vs. an abnomal cuent. Nomal Cuent The cuent should amp up to the final level in a single stage and then plateau. The plateau level is detemined by the sepaation voltage, which is set in the sepaation method (Fag-3) and is maintained thoughout the sepaation. The final level should be appoximately 7-11 µa. The cuent pofile should look simila to the figue below. 6 GenomeLab GeXP

11 Geneal Diagnostic Guidelines Evaluating the Cuent Pofiles Figue 1.4 Nomal Capillay Cuent Pofile and coesponding Raw Data Abnomal Cuent A numbe of chaacteistics can be used to detemine a cuent failue fo GeXP. The list below epesents the most commonly obseved chaacteistics fo a failed cuent: amps in two o moe stages does not maintain the sepaation cuent at a steady level a change in cuent level of moe than 10% A cuent failue, as shown in Figue 1.5, will cause the GeXP fagments to do the following: sepaate abnomally with delayed peaks (Figue 1.5) have educed peak esolution have low signal IMPORTANT If a capillay has cuent failue, do not use data fom these wells because the peak aea calculation is affected by abnomal cuent. Figue 1.5 Abnomal Capillay Cuent Pofile and coesponding Raw Data Toubleshooting Guide 7

12 Geneal Diagnostic Guidelines Evaluating the Cuent Pofiles Causes of Cuent Abnomalities Cuent pofile abnomalities can be caused by the following: a bubble in the manifold no sepaation buffe impuities o debis in the sample See "Pefoming Instument Diagnostics" on page 9, fo instuctions on how to veify the cause of the poblem. 8 GenomeLab GeXP

13 Instumentation & Chemisty Oveview 2Instumentation & Chemisty Oveview Issues encounteed with the GeXP pocess can be divided into two aeas: instumentation and chemisty. A systematic appoach to toubleshooting is descibed below, to enable the use to effectively diagnose a poblem with the GeXP pocess. Fist, use the aw data, cuent pofile and analyzed data to detemine whethe the issue is caused by one of the following: GeXP instument GeXP chemisty GeXP instument and chemisty Initial Indications If the size standad peaks and/o cuent ae abnomal, then begin by toubleshooting the instument. See "Pefoming Instument Diagnostics" on page 9. If the size standad peaks and cuent ae nomal, then begin by toubleshooting the Chemisty. See "Testing the Chemisty" on page 11. If you ae unsue whee to stat, begin by toubleshooting the GeXP instument. 2.1 Pefoming Instument Diagnostics The sepaation of Size Standad-400, in the absence of GeXP eaction poducts, is pefomed to test the integity of the GeXP instument and the vaious eagents used by the system. These eagents include: sepaation gel sample loading solution capillay aay Running Size Standad Thaw Size Standad-400 (PN ) and Sample Loading Solution (PN ) at oom tempeatue. NOTE It is ecommended that the lot numbes of all consumables be ecoded duing toubleshooting. 2. Combine 13 µl of ss-400 with 1027 µl of SLS in a micocentifuge tube and mix. IMPORTANT Use only Beckman Coulte SLS. Do not substitute SLS with fomamide fom a thid-paty. IMPORTANT Use non-baie pipette tips when pipetting SLS. 3. Pipette 40 µl of the ss SLS mixtue into each of 24 wells of a sample plate (thee ows total). 4. Place a dop of Beckman Coulte mineal oil (PN ) ove the ss SLS mixtue in each well. 5. Add Sepaation Buffe to each coesponding well of a buffe tay, filling each well about ¾ full (250 µl). 6. Pefom Sample Plate Setup. Toubleshooting Guide 9

14 Instumentation & Chemisty Pefoming Instument Diagnostics 7. Select the Fag-3 sepaation method fo all thee ows. 8. Edit the Default Fagment Analysis Paametes so that AE-Ve1 dye mobility calibation is selected in the Advanced option of Analysis Paametes. 9. Select the edited method fo automatic analysis of each well. 10. Run the samples accoding to the standad GeXP pocedues. 11. Review the data as descibed in "Geneal Diagnostic Guidelines" on page 3. Intepeting the Results The Size Standad-400 povides acceptable esults by meeting the following citeia: all size standads peaks ae pesent and called the peaks ae well esolved the cuent is nomal If this is the case, the GeXP instument and eagents ae fine and futhe diagnostics of the chemisty ae needed. If any of the above citeia ae not met, ty testing the system consumables such as the sepaation gel, SLS, the capillay aay and anothe lot of the Size Standad-400. The individual components should be tested in sepaate expeiments to identify the faulty component. If cuent failue is the poblem, poceed with using the Sequencing Test Sample to assess the instument and consumable eagents. See "Running the Sequencing Test Sample" below fo moe infomation. Running the Sequencing Test Sample The Sequencing Test Sample is used to test the integity of the GeXP instument and the consumable eagents used by the system, paticulaly when thee is a poblem with sepaation cuent. 1. Thaw thee vials of Sequencing Test Sample (PN ) at oom tempeatue. NOTE It is ecommended that the lot numbes of all consumables be ecoded duing toubleshooting. 2. Pipette 40 µl of Sequencing Test Sample diectly into each of 24 wells of a sample plate (thee ows total). IMPORTANT Use non-baie pipette tips when pipetting Sequencing Test Sample, which contains SLS. 3. Place a dop of Beckman Coulte mineal oil ove the Sequencing Test Sample in each well. 4. Add Sepaation Buffe to each coesponding well of a buffe tay, filling each well about ¾ full (250 µl). 5. Pefom sample plate Setup and select the Seq-Test sepaation method. 6. Run the samples accoding to the standad GeXP pocedues. 7. Assess the cuent pofile of data obtained with Sequencing Test Sample in the Sequencing Analysis module. Click File Open Sample Data tab then select sample files fom the appopiate poject and click OK. 8. Review the cuent pofile. The cuent should amp in one stage and plateau at 5-9 µa. The cuent pofile should look simila to the cuent in the example in the figue below. 10 GenomeLab GeXP

15 Instumentation & Chemisty Testing the Chemisty Figue 2.1 Sequencing Cuent Intepeting the Results The Sequencing Test Sample povides acceptable esults by meeting the following citeia: good aw data signal nomal cuent pofile meets the system specifications of 98% accuacy at 700 bases, when the LFR-1 sepaation method is used If this is the case, the GeXP instument and eagents ae fine and futhe diagnostics of the chemisty ae needed. NOTE Fo moe infomation on Sequence Analysis and toubleshooting poblems with sepaation cuent, efe to the Sequence Analysis Toubleshooting Guide (390216) which can be downloaded fom If the Sequencing Test Sample does not yield an acceptable esult ty testing the othe system consumables such as the sepaation gel, the capillay aay and anothe lot of the Sequencing Test Sample. The individual components should be tested in sepaate expeiments to identify the faulty component. If an acceptable esult is still not achieved afte substituting all new components contact you Beckman Coulte Field Sevice Repesentative fo sevice on the GeXP system. 2.2 Testing the Chemisty Once it is detemined that the GenomeLab GeXP instument is functioning popely, then poceed with toubleshooting the following components of GeXP chemisty: GeXP Stat Kit expeimental pocess thid-paty eagents expeimental RNA custom multiplex pimes Testing the GeXP Stat Kit and Expeimental Pocess Test the components of the GeXP Stat Kit (A21019) and the use s expeimental pocess by pefoming standad GeXP eactions with contol eagents. Toubleshooting Guide 11

16 Instumentation & Chemisty Testing Thid-Paty Poducts 1. Pefom GeXP eactions with Contol RNA and a set of GeXP multiplex pimes peviously demonstated to geneate size-specific amplicons, such as those povided in the GenomeLab GeXP multiplex gene set kits fom Beckman Coulte, as listed below: Human RefeencePlex (A54651) Human MetastasisPlex (A32712) Human Beast CancePlex (A32700) Rat MultitoxPlex (A21066) 2. Analyze the eactions with the GeXP instument and examine the analyzed esults. Intepeting the Results If all of the multiplex peaks and one KAN peak ae pesent, this confims the functional quality of the GeXP Stat Kit eagents and thid-paty-supplied mateials. These esults also confim that the use s expeimental pocess is satisfactoy. These esults do not confim the functional quality of any custom-designed multiplex pimes and RNA template. See "Testing the Expeimental RNA and Custom Multiplex Pime" on page 13 and continue in the toubleshooting pocess with these eagents. If the test does not yield all multiplex peaks and one KAN peak, with good aw data signal and nomal cuent pofiles, pefom the test again using a new GeXP Stat Kit. Failue to yield all multiplex peaks and one KAN peak with a second kit would indicate that thid-paty eagents o the expeimental pocess ae suspect. See "Examining the Expeimental Pocess" below and "Testing Thid-Paty Poducts" on page 12. Examining the Expeimental Pocess The following ae items to eview as pat of a successful GeXP Expeimental Pocess: Reagent handling and stoage RNA handling and stoage Accuate pipetting Potocol Steps See the GeXP Chemisty Potocol (A29143) fo additional instuctions on pefoming this pocess. 2.3 Testing Thid-Paty Poducts Some eagents and plasticwae, supplied by thid-paties can have a negative impact on the GeXP RT-PCR eaction, aw data signal and the cuent pofile. Thid-paty poducts includes the chemicals and plasticwae used fo suspension and dilution of RNA and pimes, the RT-PCR eactions and the pe-dilution of the PCR eaction poducts. Thid-Paty Poducts: Do s & Don ts Use high-quality, nuclease-fee plasticwae. Suspend pime multiplexes in 10 mm Tis-HCl, ph GenomeLab GeXP

17 Instumentation & Chemisty Testing Thid-Paty Poducts Stoe stock solutions of RNA samples in a buffeed solution, such as The RNA Stoage Solution (Ambion PN AM7000). Stoe stock solutions of RNA in small aliquots at -80 C to peseve RNA integity. Suspend woking concentations of RNA in nuclease-fee, non-depc teated wate, as supplied in the GenomeLab GeXP Stat Kit o fom USB (PN 71786) o Invitogen (PN ). IMPORTANT Do not use DEPC-teated wate o plasticwae with the GeXP pocess. Residual DEPC can inhibit the PCR eaction and esult in low signal stength and a high baseline in the electopheogam. Use aeosol-esistant baie pipette tips with a dedicated set of pipettes fo the setup of evese tansciption (RT) and PCR eactions to pevent coss-contamination of samples. Do not allow amplified poduct to ente the aea of RT and PCR eaction setup (amplicon-fee zone). Use non-baie pipette tips and a sepaate set of pipettes, when handling the PCR poducts, Sample Loading Solution (SLS) and Size Standad-400 in a PCR amplicon zone. The SLS eagent may dislodge filte paticles of aeosol-esistant baie tip into the sample and this contamination can cause cuent failue in the GeXP System. Pefom pe-dilution of the PCR eaction with 10 mm Tis-HCl, ph 8.0 Intepeting the Results The common symptoms fo poo quality eagents o the wong eagent concentation ae low aw data signal, high baseline and eatic cuent pofiles. If the GenomeLab GeXP multiplex gene set kit eactions with Contol RNA as outlined in "Testing the GeXP Stat Kit and Expeimental Pocess" on page 11, yield all the multiplex peaks and one KAN peak and the aw data signal baseline and cuent pofiles look good, then thid-paty eagents ae satisfactoy. If the contol multiplex eactions did not yield all of the multiplex peaks o the aw data o cuent pofile wee abnomal, then eplace each thid-paty eagent in a systematic appoach (one at a time) to identify the faulty eagent. Pefom GeXP contol eactions as descibed in "Testing the GeXP Stat Kit and Expeimental Pocess" on page 11, and analyze them with the GenomeLab GeXP. If the expeimental RNA and custom multiplex pimes still do not yield acceptable esults, and a specific thid-paty poduct was not identified as the cause, poceed to the next section. Testing the Expeimental RNA and Custom Multiplex Pime Afte confiming that the GenomeLab GeXP multiplex gene set kit and Contol RNA function coectly with the GeXP instument, test the expeimental RNA and custom multiplex pimes. Test the eagents independently. This is the most efficient means of identifying the souce of the poblem. Testing the Expeimental RNA NOTE Use high quality expeimental RNA that has a 28S/18S atio geate than 1.0 with GeXP. Confim that the ibosomal RNA 28S and 18S bands ae eadily visible and pedominant when analyzed on an agaose gel. Use a set of contol GeXP multiplex pimes that ae known to poduce size-specific amplicons fom the souce of expeimental RNA, in ode to test the functional integity of Toubleshooting Guide 13

18 Instumentation & Chemisty Testing Thid-Paty Poducts the expeimental RNA. Fo example, expeimental human RNA sample(s) can be tested with multiplex pimes fom the GenomeLab GeXP Human RefeencePlex Kit (PN A54651) which contains 24 sets of pimes that taget human housekeeping and othe efeence genes. See "Intepeting the Results" on page 14 fo moe infomation. Testing the Custom Multiplex Pimes NOTE Ode custom multiplex pimes with the univesal tag sequences fused to the gene-specific sequence. The oligos should be of standad desalted, depotected pocessing. Duing the initial evaluation of the multiplex, the chimeic pimes must be evaluated fo thei ability to poduce the expected size amplicon with Contol RNA (RNA template known to contain the taget tanscipt). Fo example, the Human Refeence Contol RNA fom the GenomeLab GeXP Human RefeencePlex Kit (PN A54651) can be used as a template to test custom multiplex pimes that taget human tanscipts. NOTE While the Human Refeence Contol RNA povides boad gene coveage, not all the gene tanscipts ae necessaily pesent. It is highly advisable to have a thoough undestanding of the gene expession pattens fo the tanscipts detected by the custom multiplex pimes. Develop a custom Contol RNA by mixing RNA fom seveal souces to achieve full tanscipt epesentation. Geneally, this mixtue will contain 50% of nomal and 50% teated RNA. NOTE If moe than one teatment is being studied, combine the teatments so that each one is equally epesented in the 50% teated RNA. Evaluate evese pimes in a multiplex context. Evaluate fowad pimes in both multiplex and singlet eactions. See the GeXP Chemisty Potocol (PN A29143) fo moe infomation. Intepeting the Results If all of the multiplex peaks and one KAN peak ae pesent, with no significant undesigned peaks (UDPs), the functional quality of the expeimental RNA and custom multiplex pimes ae confimed. See "Refeence Tables" on page 17 fo moe infomation on UDPs. If a eaction containing expeimental RNA and contol multiplex pimes yields a KAN peak, but no multiplex peaks o multiplex peaks with low signal, then it is likely that the quality of the RNA is poo. Re-evaluate the RNA souce o puification pocess to yield highe quality RNA. If a eaction containing custom multiplex pimes and Contol RNA yields a KAN peak, but is missing one o moe peaks in the multiplex, then it is likely that edesign is equied fo the pime(s) of the missing peak(s). See "Multiplex Pime Design" on page 17 fo moe infomation on toubleshooting this issue. If a eaction containing custom multiplex pimes and Contol RNA yields a KAN peak and all the multiplex peaks, but has significant UDPs, then pime edesign is necessay. See "Multiplex Pime Design" on page 17 fo moe infomation on toubleshooting this issue. If no eaction yields a KAN peak (positive contol), then see "Testing the GeXP Stat Kit and Expeimental Pocess" on page 11 to e-evaluate the stat kit components and expeimental pocess. 14 GenomeLab GeXP

19 Instumentation & Chemisty Testing Thid-Paty Poducts By using the peviously descibed pocess, you should have been able to identify many of the most common causes of GeXP poblems associated with instumentation and chemisty in capillay electophoesis-based gene expession pofiling. Refe to the "Refeence Tables" on page 17 o the "Questions & Answes" on page 31 fo additional causes and coective actions fo toubleshooting puposes. Toubleshooting Guide 15

20 Instumentation & Chemisty Testing Thid-Paty Poducts 16 GenomeLab GeXP

21 Refeence Tables Multiplex Pime Design 3Refeence Tables The following tables list issues that might be encounteed while analyzing GeXP multiplex eactions with the GenomeLab GeXP Genetic Analysis System. Multiplex Pime Design on page 17 GeXP Chemisty on page 20 GeXP Sepaation and Fagment Analysis on page 25 expess Pofile and Analysis on page 28 Fo specific solutions o examples, look fo efeences to sample figues in Appendix B. 3.1 Multiplex Pime Design Issue Cause Solution Some of the accession numbes o genes enteed into expess Multiplex ae not incopoated into the multiplex Incoect accession numbe fomat The accession numbe is not ecognized by expess Pofile. Use Vebose Mode to identify which genes ae not incopoated by executing the multiplex. See "Toubleshooting Multiplex Design with Vebose Mode" on page 37. Incoect gene sequence fomat Use the coect accession numbe fomat, including undescoes (_), if pesent. The gene sequence is not ecognized by expess Pofile. Use the FASTA function to veify that the accession numbe efes to nucleotide sequence, not potein sequence. expess Pofile only ecognizes the chaactes: A,C,G, T and N fo gene sequence. If the gene sequence contains othe IUPAC code, ceate a popietay sequence that excludes this egion of the sequence. IMPORTANT Do not use a sequence with any othe IUPAC code (i.e. R, Y, S, W). Altenatively, seach GenBank fo anothe accession numbe efeing to the same gene with sequence containing only A,C,G,T and N. Toubleshooting Guide 17

22 Refeence Tables Multiplex Pime Design Issue Cause Solution Pimes enteed by the Administato in Manage Pimes ae not displayed in the Pimes field of the Multiplex Designe Multiple peaks appea fo one set of pimes in evese singlet + fowad singlet eaction Undesigned peak(s) (UDP) is pesent in singlet o multiplex eactions One o moe designed gene peaks is absent fom multiplex pofile but KAN peak is pesent Softwae will not show the pimes unless they ae set as Refeence Pimes mrna isofoms o homologous sequence Non-specific amplification Poo pime design RNA sample does not contain the tanscipt As Administato, select Save as Refeence Pime in Manage Pimes to make a pime set globally available. See Figue B.1 on page 41 The gene sequence used fo pime design may have led to amplification of altenative tanscipts (isofoms) o a homologous sequence in anothe gene. Be sue to choose mrna o cdna, and not genomic DNA, sequence files fo pime design. BLAST designed pime sequences to detemine if moe than one poduct will be amplified. Use sequence alignment softwae to find a egion of the gene that does not shae homology with any othe gene. Specifically taget that egion in the Pime Design option o ceate a popietay gene sequence with the Manage Genes function in the Administato module. Fo tanscipt vaiants, design pimes to bidge a specific exon-exon junction such that a specific tanscipt is amplified. See "Designing Pimes to Detect Altenative Tanscipts" on page 38. If a UDP migates within 3nt of a designed peak in the multiplex o affects the calculation of a designed gene peak aea, then pefom singlet eactions to identify the pime(s) causing the UDP. See Figue B.2 on page 42. Redesign the offending pime by tageting a diffeent sequence o move the affected amplicon(s) to a diffeent location in plex (diffeent size), away fom the UDP. Pefom singlet eactions to detemine if each set of pimes amplifies the expected fagment size. Redesign pimes fo those genes that do not yield an acceptable singlet pofile. Use an RNA sample that contains the tanscipt 18 GenomeLab GeXP

23 Refeence Tables Multiplex Pime Design Issue Cause Solution A gene peak disappeas fom the multiplex pofile when it is expected to be pesent in the RNA sample Low o no signal fo a paticula peak in a multiplex eaction with RNA expected to contain the tanscipt, using pimes that geneate a peak in a singlet eaction. A paticula amplicon has two o moe shouldes o stutte peaks. Significantly highe %CV in biological eplicates compaed to technical eplicates Poo pime design Pime intefeence Repeats in amplicon sequence cause polymease slippage See Figue B.3 on page 42 Thee is a SNP in the 3' end of pime sequence If the peak is pesent in some wells, but not othes pepaed fom the same RNA sample and Maste Mix eagents (RT, PCR), then the pime may not have good binding specificity. Polymophism(s) in the pime sequence, especially at the 3' end, may esult in amplification fom one sample RNA but not anothe. Redesign the pime set to a bette egion of the gene (not too close to eithe the 5' o 3' end of the tanscipt, no epeat sequences, no GC ich egions, no homologous egions, no polymophisms). It is most efficient to edesign the affected pime(s). Howeve, if it is necessay to detemine which pime is the intefeing pime, pefom duplex eactions containing the affected pime with each of the othe pime sets in the multiplex, then edesign the intefeing pime. Check the designed amplicon fo epeat sequences. Redesign the pimes to a egion of the gene that does not contain epeat sequences. Check the pimes fo single nucleotide polymophisms (SNPs). Redesign the pime if any SNPs ae pesent. SNPs can have pofound impact on the piming efficiency depending on how much of the instability is intoduced. A biological sample with the pefect match to the pimes will geneate a highe signal than a sample with a mismatch due to the pesence of SNPs. This will esult in high %CV in biological eplicates, but not technical eplicates. Biological eplicates geneally have slightly highe %CV than technical eplicates, due to the natual vaiation between oganisms o the vaiation that was intoduced duing sample pepaation. Toubleshooting Guide 19

24 Refeence Tables GeXP Chemisty 3.2 GeXP Chemisty Issue Cause Solution A vaiation in multiplex quantitative esults is seen fom build-to-build of custom pime plexes fo the same RNA sample Multiple designed gene peaks ae pesent in evese multiplex + fowad singlet eactions The multiplex fomulation has changed Pime contamination Once optimized, do not change the multiplex fomulation (pime concentations). Caefully assemble each multiplex build fo consistency. Remake the multiplex consistent with the optimized fomula. Make sue to use the same pime plex fomulation thoughout a study. The evese pime stocks o evese pime multiplex is contaminated with fowad pimes. Altenatively, fowad pime stocks ae coss-contaminated with othe fowad pimes. Decontaminate the lab bench, pipettes, etc. with a nucleic acid-destoying solution Use fesh eagents and emake pime stocks and/o evese multiplex Use aeosol-baie filte tips fo making multiplexes and assembling RT and PCR eactions 20 GenomeLab GeXP

25 Refeence Tables GeXP Chemisty Issue Cause Solution Low signal Pime Design See this topic in "Multiplex Pime Design" on page 17. DEPC intefeence IMPORTANT Do not use DEPC-teated wate fo GeXP as esidual DEPC can intefee with PCR amplification. Pime degadation Pime quality Reagents - expied and/o impope stoage/handling RNA template - quality, quantity Themal cycle Use Nuclease-fee wate (USB o Invitogen ) when making Resuspension Buffe (10 mm Tis-HCl, ph 8) fo pime plexes. Use 10mM Tis-HCl, ph 8 (Resuspension Buffe) fo making pime plexes. Stoe multiplex pimes at -20 C. Pimes can contain esidual amounts of oganic solvent. Ode new pimes fom well-espected oligo vendos, such as Integated DNA Technologies (IDT). Check eagent stoage conditions and expiation dates. All kit components, except RNA, should be stoed at -20 C. Avoid excessive feeze-thaw cycles. Contol RNA and KAN RNA should be aliquoted into single use volumes afte the fist thaw and always stoed at -80 C. Use fesh eagents that have been popely stoed. Votex the 5x RT Buffe containing DTT and the 25 mm MgCl 2 to dissolve any pecipitant befoe use. Veify that the RNA is of high quality and adequate quantity. Ribosomal RNA 28S and 18S bands should be pominent on agaose gel and 28S/18S atio > 1.0. Recheck calculations fo amount of input total RNA ( ng is ecommended). Incease the amount of RNA template used. Check themal cycle calibation and potocol cycling tempeatues. Raise the PCR extension tempeatue to 70 C to ovecome vaiation in themal cycle wells. Use a themal cycle with a heated lid to pevent evapoation and veify that the lid tempeatue is the same tempeatue as the incubation chambe. Toubleshooting Guide 21

26 Refeence Tables GeXP Chemisty Issue Cause Solution Low Signal Sample degadation o Photobleach RT eactions can be stoed at -20 C fo up to one month. Do not expose 5x PCR buffe o PCR eactions to light fo an extended peiod of time. Stoe PCR eactions at -20 C in the dak (wapped in foil) fo up to one month. Diluted PCR sample may degade faste in unbuffeed wate. It is ecommended to use fesh 10mM Tis-HCl, ph 8 fo pe-dilutions of PCR eactions. High Signal (peak height is geate than 120,000 RFU in analyzed data fo one o moe fagments) Capillay electophoesis Revese pime concentation is too high See Figue B.4 on page 43 Too much RNA Too much PCR poduct loaded See this topic in "GeXP Sepaation and Fagment Analysis" on page 25. Attenuate the evese pime(s) by educing the concentation in the evese multiplex Reduce the amount of RNA. See this topic in "GeXP Sepaation and Fagment Analysis" on page GenomeLab GeXP

27 Refeence Tables GeXP Chemisty Issue Cause Solution One o moe designed gene peaks ae absent fom multiplex pofile but KAN peak is pesent Poo pime design See this topic in "Multiplex Pime Design" on page 17. RNA The RNA sample may not contain the tanscipt(s) o the gene is downegulated in that sample. Use moe RNA in the evese tansciption eaction (up to 100ng pe eaction) Use an RNA souce known to contain the gene tanscipt of inteest to validate the pime design Combine RNA samples/souces such that all genes in multiplex ae epesented at a elatively modeate detectable level and use this RNA as a positive contol fo gene detection and multiplex optimization RNA may be degaded. Veify the RNA quality (Ribosomal RNA 28S/18S atio should be > 1.0). Stoe stock RNA samples in The RNA Stoage Solution (Ambion AM#7000), at -80. Aliquot to woking volumes, and thaw aliquots only once to ensue RNA integity. Pime concentation Incease evese pime up to 150 nm pe eaction (1.5 µm in the evese multiplex) fo each low expesse. Peaks ae pesent in RT minus eactions. Genomic DNA contamination See Figue B.6 on page 44 Amplification contaminated with anothe DNA template o DNA amplicons Well-to-well contamination Additionally, deceasing the high signal of high expesses though attenuation may bing up the signal of low expesses. Teat RNA with RNase-fee DNase duing RNA puification. Use aeosol-esistant baie tips duing RT and PCR eaction pepaation to minimize contamination fom extenal souces. Use fesh tips fo each step in pepaing the GeXP eactions Sepaate pe- and post-pcr wok aeas. Do not bing amplified poduct into pe-pcr aea. Contamination acoss wells can occu duing set up fo PCR o sepaation, especially with single channel pipetting. Use multichannel pipette to educe pipetting eo and contamination. Toubleshooting Guide 23

28 Refeence Tables GeXP Chemisty Issue Cause Solution Low o no signal fo a paticula peak in a multiplex eaction using pimes that wee validated in a singlet eaction Undesigned peak(s) (UDP) is pesent in multiplex eactions Sloping gene expession pofile o Smalle fagments have much highe signal than lage fagments. Pime intefeence See this topic in "Multiplex Pime Design" on page 17. Non-specific amplification Signal dop-off See Figue B.7 on page 44 See this topic in "Multiplex Pime Design" on page 17. Themal cycle tempeatue fluctuation duing PCR extension leads to a dispopotionate amplification of shote fagments ove long fagments, called signal dop-off. This commonly occus in the edge wells of a themal cycle that is not pefoming well. Repai and/o calibate the themal cycle Use the cente wells of a themal cycle Altenatively, optimize the extension tempeatue; geneally by aising the extension tempeatue by a degee o two Unbalanced pofile o Refeence (housekeeping) genes ae not geneating the expected high peaks elative to othe genes in the plex. Multiplex is not optimized See Figue B.4 on page 43 IMPORTANT Signal dop-off can lead to high %CV in analyzed data and esults of these wells should not be used fo data analysis. The wong DNA Polymease was used. Beckman Coulte has validated ThemoStat DNA Polymease (A25395) fo use with the GeXP System. Attenuate the concentation of evese pimes fo high signal peaks and high expesses. The pupose of attenuation is to bing down the peak height of high expesses to the level of modeate expesses in the same plex. It is an appoach that balances elative signal stength in the optimal detection ange. Select a modeate expesse peak in a modeate signal ange (10,000-50,000 RFU) as efeence upon which the optimal attenuation facto fo high expesses will be decided. Then bing down the high expesse peaks to the level of the modeate expesse efeence peak by educing evese pime concentation. It is not necessay, no coect, to conside absolute peak height fo this appoach. Incease the concentation of evese pimes fo low expesses up to 150 nm pe eaction to incease signal stength elative to the modeate expesse gene(s). 24 GenomeLab GeXP

29 Refeence Tables GeXP Sepaation and Fagment Analysis 3.3 GeXP Sepaation and Fagment Analysis Issue Cause Solution Cuent failue Low signal o no signal Ai bubble in the manifold No sepaation buffe o excessive sepaation buffe Poo injection due to excess salt Pefom extensive puging of the GeXP instument, with fesh sepaation gel befoe each un. Pefom a Manifold Puge thee times with 0.4 ml gel. Then pefom thee capillay fills. Reun PCR samples on GeXP. Add the ecommended amount of sepaation buffe to each well of the buffe plate that coesponds to a filled well of the sample plate. Keep the buffe evapoation cove ove the buffe plate when installed on instument. Too much salt in a sample can lead to poo injection of amplicons which leads to low signal. High Signal (peak height is >120,000 RFU in analyzed data fo one o moe fagments) Additional peaks fom diffeent dye-channels ae pesent in the data Unesolved fagment in a paticula size ange is pesent in standad (STD) and no template contol (NTC) eactions. Use fesh 10 mm Tis-HCl, ph 8 to pe-dilute PCR eactions, if necessay, befoe adding the diluted sample to Sample Loading Solution with Size Standad 400. Chemisty See this topic in "GeXP Chemisty" on page 20. Too much RNA Too much PCR sample was used fo loading See Figue B.5 on page 43 Too much PCR sample was used fo loading See Figue B.8 on page 45 Potein-nucleic acid complex See Figue B.9 on page 46 Reduce the amount of RNA used in the RT eaction. Pepae seial dilutions (1:5, 1:10, 1:20) in 10 mm Tis HCl, ph 8 of the PCR poducts. Mix 1 ul of the pe-diluted sample to Sample Loading Solution with Size Standad 400 and test each dilution in all capillaies to find the best dilution fo analysis. The linea ange of detection lies between ,000 RFU in analyzed data. It is ecommended that data outside this ange not be used fo analysis. If the peak height (signal) is too high (>120,000 RFU), a small peak may be obseved in an altenate dye channel eithe diectly adjacent to o undeneath the ove-anged peak. Decease the amount of sample loaded on the instument o pe-dilute the PCR poduct in 10mM Tis-HCl, ph 8. A non-specific, potein-nucleic acid complex will migate in a paticula size egion of electopheogam. This complex is of consistent size, specific to a multiplex and does not affect analysis unless it ovelaps with a designed peak. Redesign the pimes of any gene peaks that comigate with the complex. Toubleshooting Guide 25

30 Refeence Tables GeXP Sepaation and Fagment Analysis Issue Cause Solution Valid peaks wee not analyzed o called in Fagment Analysis Size shift in designed gene peaks and/o KAN peak The peak fo a low expesse o uninduced gene was not detected. Too many small, undesigned peaks called Analysis paametes Exclusion filte See Figue B.10 on page 47. Analysis paametes Wong size standad Analysis paametes See Figue B.12 on page 48 and Figue B.13 on page 48 Instument Sensitivity The wong analysis paametes can lead to poo analysis. Use the Default GeXP Analysis Paametes: Slope Theshold = 10%, Relative Peak Height Theshold = 1%, Confidence level = 95%, SizeStd 400, Cubic Model, Dye Mobility Calibation ON = PA ve.1, Calculated Dye Specta. The Slope and Peak Height Thesholds can be educed to pick up small peaks. An exclusion filte in the Fagment List tab of Fagment Analysis module may be active. Exclusion filtes, specific to each multiplex, need to be adjusted and saved fo each GeXP instument to account fo vaiation in sepaation. Adjust sizes of exclusion filte to include valid peaks with a bin width of appoximately 2 nucleotides. See Figue B.11 on page 47. Use the Default GeXP Analysis Paametes: Slope Theshold = 10%, Relative Peak Height Theshold = 1%, Confidence level = 95%, SizeStd 400, Cubic Model, Dye Mobility Calibation ON = PA ve.1, Calculated Dye Specta. Veify which Size Standad chemisty was used. GeXP Analysis Paametes ae designed to be used with Size Standad-400. If Size Standad-600 was used, the fagment sizing will be diffeent fo samples analyzed with Default GeXP Analysis Paametes. Edit the GeXP Analysis Paametes so that Size Standad-600 and Quatic model ae selected and then eanalyze the data. Save this evised Analysis Paamete with a new name (i.e. GeXPss600). When GeXP analysis paametes ae loweed to Slope Theshold = 1 and Peak Height Theshold = 0, nealy evey peak, can be detected. If a vey small peak (~370 RFU) with the exact same fagment size as the induced gene is detected, it can be teated as eal peak in the uninduced sample. It is best if this vey small, uninduced peak esides in an aea with a clean baseline (no UDPs o excessive noise) of the multiplex pofile so the peak call is accuate. See Figue B.13 on page 48. Exclude the small, insignificant peaks by establishing an exclusion filte in Fagment List of Fagment Analysis so that the peaks below a paticula peak height will not be called. Altenatively, use the exclusion filte to exclude unwanted peaks fom bins of paticula width. See Figue B.11 on page GenomeLab GeXP

31 Refeence Tables GeXP Sepaation and Fagment Analysis Issue Cause Solution Analysis eo Ove-ange data The data is above the instument's linea ange of detection and the softwae cannot analyze the data popely. Any ove-ange data should not be used fo expession analysis. Split peaks with extemely high signals in analyzed data Ove-ange data See Figue B.14 on page 49 Dilute PCR samples in 10 mm Tis-HCl, ph 8 to bing data into the linea ange; below 120,000 RFU in analyzed data. Attenuate high expesses if necessay, to balance the pofile. Check the aw data to confim the esults. Do not use ove-ange data fo expession analysis. Dilute PCR sample in 10 mm Tis-HCl, ph 8 to bing data in linea ange of detection, less than 120,000 RFU in analyzed data. Inconsistent fagment sizes o unusual peak shapes o low signal Attenuate the high expesses if necessay to balance pofile. Gel life exceeded Check the on-boad gel life. Gel life is veified fo 72 hous on the instument. Capillay aay life exceeded Failue to maintain pope capillay sepaation tempeatue Check capillay aay life. Capillay aay life has been veified fo 100 uns o 30 days on instument, whicheve comes fist. Check the un log fo veification of sepaation tempeatue. Reun the samples. If the poblem continues, contact you Beckman Coulte Field Sevice Repesentative. Toubleshooting Guide 27

32 Refeence Tables expess Pofile and Analysis 3.4 expess Pofile and Analysis Issue Cause Solution Eo message stating connection timed out o not detected Intenet connection Intenet connection was lost by expess Pofile. The system must be estated with the intenet connected. Exit the pogam and then e-launch GeXP expess Pofile. NOTE The connection to NCBI will not wok unless the expess Pofile can see the intenet on pot 80. Fozen sceen o missing gene infomation duing peak binning Data fom Fagment Analysis won't impot into expess Analyze An undesigned peak (UDP) is close to a designed gene peak in the electopheogam Coupt TDF file Wong expot fomat Binning and Peak Detection To test this, open the intenet bowse and bowse to If the Beckman Coulte, Inc. website does not open, contact you netwok administato. File couption is likely to occu duing the impot of the TDF file if the Impot button is e-clicked befoe the impot is completed. See "Repaiing a Coupt TDF File" on page 37 fo moe infomation. Expot fagments using the Tansfe Fagments fo GeXP (not Expot Results o Expot Fagments/Genotypes) option fom Fagment Analysis. Set the bin size and width to exclude any UDP that has a highe signal than the designed peak signal. Only tallest peak within a bin will be selected fo quantitative analysis and nomalization. As long as the signal of the UDP is consistently lowe than that of the designed peak, acoss the entie plate, the designed peak will be selected. 28 GenomeLab GeXP

33 Refeence Tables expess Pofile and Analysis Issue Cause Solution High %CV Sample size Pefom at least thee technical eplicates pe RNA sample. Sample type Refeence Gene Geneally, moe technical eplicates of the same RNA sample o RT eaction educes the %CV. A small numbe of biological eplicates can lead to a high %CV due to inheent divesity between oganisms. Low expesse genes tend to have highe %CV. Review the choice of efeence gene to veify that elative expession of this gene is constant acoss all samples. If the efeence gene expession fluctuates geatly between samples, this could influence the %CV. Choose a efeence gene that has constant expession unde all conditions that will be examined in the study. The GenomeLab Human RefeencePlex Kit (A54651) is a validated assay designed to detemine the best efeences gene fo human RNA samples. NOTE Nomally, the KAN peak signal is usually out of ange and should theefo not be used as a efeence gene fo nomalization of expeimental genes. If it is desied to use KAN as a efeence gene fo nomalization, the amount of KAN RNA needs to be educed so that the peak height of KAN is simila to that of the median-expesses in the multiplex. Signal dop-off See this topic in "GeXP Chemisty" on page 20. SNPs in the pime(s) See this topic in "Multiplex Pime Design" on page 17. Toubleshooting Guide 29

34 Refeence Tables expess Pofile and Analysis Issue Cause Solution Thid-paty softwae fo gene expession analysis Data analysis IMPORTANT Do not install thid-paty softwae on the expess Pofile contolle fo futhe analysis of gene expession data. The following analyses can be pefomed on GeXP data, using Micosoft Excel: multiple efeence gene nomalization calculation of fold-change statistical analysis Disuption in intenet connection, geneal malfunction of softwae Thid-paty softwae, Micosoft Windows updates Additional softwae pogams fo use with GeXP-geneated data ae GeNom, DecisionSite, PatekGS and Pism. Visit the websites below fo infomation on these softwae pogams. Thid-paty softwae infomation: Although they ae impotant to the Windows softwae secuity capabilities, Micosoft Updates should not be pefomed on these contolles. This default setting is tuned off. Contact you Beckman Coulte Technical Suppot Repesentative fo moe infomation. 30 GenomeLab GeXP

35 Questions & Answes expess Pofile 4Questions & Answes 4.1 expess Pofile Question Is it okay to install thid-paty softwae on my expess Pofile Contolle? No. Answe The contolle must be used as it is configued. Can I manually install Micosoft Windows updates? Can I delete unused pimes, genes o multiplexes fom expess Pofile? IMPORTANT Do not install thid-paty softwae o additional netwok cads on the expess Pofile Contolle. No. Although they ae impotant to the Windows softwae secuity capabilities, Micosoft Updates should not be pefomed on these contolles. This default setting is tuned off. Contact you Beckman Coulte Technical Suppot Repesentative fo moe infomation. No. These ae files that ae designed to be shaed by multiple uses. Genes and pimes could be used in seveal multiplexes. 4.2 Multiplex Pime Design Question Can I ceate a multiplex without an intenet connection? What ae the optimal conditions to use when I manually design o edesign pimes fo use with GeXP? Answe Yes. NOTE Befoe you begin, confim that all gene sequences ae available to copy fom anothe file and paste into Manage Genes. See "Ceating a Multiplex without an Intenet Connection" on page 38. Although these ae the optimal conditions fo pimes, individual pimes within a multiplex will vay. Appoximately 20 nt in length, without univesal tag 50% G+C content Tm = 60 C (Range: C) the last 5 nucleotides at 3' end should contain at least 2 As o Ts and not contain any polymophisms The pime length can be vay fom 20 nt as long as it meets the othe conditions. Toubleshooting Guide 31

36 Questions & Answes GeXP Chemisty and Fagment Analysis Can I design pimes to detect altenative tanscipts? Can I can add o delete a pime fom a peviously designed multiplex? Yes. Fist, define the mrna isofom(s) of inteest and design pimes specifically to include o exclude exons o to bidge unique exon-exon junctions. See "Designing Pimes to Detect Altenative Tanscipts" on page 38 fo moe infomation. Yes. See "Modifying a Peviously Designed Multiplex" on page 39 fo instuctions. 4.3 GeXP Chemisty and Fagment Analysis Question What is the diffeence between the pe- and post- PCR aeas? What can happen if amplified PCR poduct is bought into the pe-pcr aea? What RNA should I use to evaluate and optimize my custom multiplex? Answe The pe-pcr aea is used to set up both the evese tansciption (RT) and PCR eactions. The post-pcr aea is whee the PCR eaction goes afte amplification. The pe-pcr aea is divided into two zones: No Template Zone: Assemble and aliquot the maste mixes Template Addition Zone: Add RNA fo the RT eaction o add cdna to the PCR eaction IMPORTANT Amplified (PCR) poduct should neve be bought into the pe-pcr aea. Use the themal cycle fo PCR in the post-pcr aea. Any aea that is exposed to amplified PCR poduct should be consideed as an aea that contains amplified template in the envionment. One symptom of PCR poduct contamination of the pe-pcr aea is peaks in the RT minus and No Template Contol eactions of GeXP. The contamination of GeXP RT-PCR sample wells with exogenous template will compomise the elative quantitation of GeXP. To clean up a pe-pcr aea contaminated with amplified poduct o othe nucleic acid template, use a 5% bleach solution o commecially available decontaminant such as DNA Zap (Ambion) o DNA AWAY (Molecula BioPoducts), to wipe down all sufaces and equipment. Initially test the multiplex on a Contol RNA that consists of a mixtue of the RNA samples that will eventually be tested individually with the multiplex (e.g. unteated + teated o nomal + disease). All the gene tanscipts must be pesent in the Contol RNA fo initial evaluation and validation of multiplex pimes. Fo optimization of the multiplex, atios of each RNA sample in the Contol RNA should eflect a modeate level of expession fo most individual genes. NOTE Levels of expession ae elative fo each gene. 32 GenomeLab GeXP

37 Questions & Answes GeXP Chemisty and Fagment Analysis Ae undesigned peaks (UDPs) in singlet eactions cause fo concen? What do I do if an undesigned peak (UDP) co-migates with a designed peak in a multiplex? What is attenuation and how will it affect my esults? If UDPs: migates at the same size as a designed peak in the multiplex affect the quantitation of a designed gene peak then edesign pimes that cause this UDP. It is best to assess the significance of a UDP in a singlet eaction when the peak height of the designed peak is below 120,000 RFU. If it is possible to detemine which pime is causing the UDP fom singlet eactions, then edesign this pime by tageting a diffeent sequence. Altenatively, the designed peak can be moved to a diffeent location in the plex by edesigning the pimes. This may simply involve moving the pime position a few nucleotides in one diection o the othe to shift the designed peak away fom the UDP. Attenuation is the pocess by which the evese pime concentation of high expesses is educed. This bings the high expesse gene signals into ange of the modeate expesses. Attenuation is used to balance the signal of the gene expession pofile within the linea ange of detection. The GeXP instument has a lowe and uppe limit in its ange of detection ( ,000 RFU in the analyzed data). Duing multiplex optimization, it is impotant to ensue that all peaks fall within that limit, ideally within a ange between 2,000-50,000 RFU. Attenuation pefomed duing the optimization of a paticula multiplex has no effect on esults, because this optimization is caied out on Contol RNA. The goal is to establish the baseline levels of expession and gene specific evese pime concentation with Contol RNA befoe testing othe RNAs. Attenuation affects only the detection of mrna of the paticula tanscipt. This is usually a high expesse, such as a housekeeping gene that needs to be bought into the linea ange of detection. The concentation of evese gene-specific pimes ae changed only duing the plex optimization stage. Once concentations fo all pimes in a evese multiplex have been optimized, the pime concentations emain fixed fo all subsequent expeiments. Thus, the elative quantitation of gene expession fo any paticula gene will emain constant in a paticula sample. Futhe the fold change in expession fo this gene between teatments is always calculated using the elative quantitation. Fo moe infomation on Attenuation, visit ou website: and obtain a copy of the Applications Infomation Bulletin A- 2049A Multiplexed, Quantitative Gene Expession Analysis fo Lettuce Seed Gemination on GenomeLab GeXP Genetic Analysis System. Toubleshooting Guide 33

38 Questions & Answes expess Analysis What is the best way to detect the peak aea of an uninduced o low expesse gene afte pime optimization? What is the best way to detect a lage fold induction with GeXP? What is the minimum and maximum elative limit in fluoescence units (RFU) fo a designed peak to emain in the linea ange of detection? Can I use the KAN peak as a efeence gene fo nomalization? To detect the peak aea of an uninduced/low expesse gene: Change the GeXP Analysis paametes in Fagment Analysis to Slope Theshold = 1 and Peak Height Theshold = 0, and eanalyze. With these settings, nealy evey peak, can be detected. If a vey small peak (~370 fu) with the exact same fagment size as the induced gene is detected, it can be teated as eal peak in the uninduced sample. Fo the most accuate peak call, it is best if this vey small, uninduced peak esides in an aea with a clean baseline (no UDPs o excessive noise) of the multiplex pofile. Induced genes will usually have a elatively low signal (small peak) in the uninduced o basal state and high signal (tall peaks) in the induced state. Attenuate the evese pime concentation and pe-dilute the PCR poducts such that uninduced samples geneate a vey small, yet detectable peak and induced samples geneate a peak within linea ange of detection ( ,000 RFU in analyzed data). Sepaate PCR eactions fom uninduced RNA samples in middle CEQ capillaies (C, D, E, F) and eactions fom induced samples in the oute capillaies (A,B, G, H). Detection of appoximately 1000-fold change in expession can be achieved with this method ,000 RFU in the analyzed data. This is the ecommended signal ange fo quantitation with GeXP. In ode to detect vaiations in gene expession fom sample to sample, design gene peaks within the ange of appoximately ,000 RFU, duing multiplex optimization. Yes, unde cetain conditions. KAN RNA is an independent template that is designed to seve as a positive, intenal contol fo the RT and PCR eactions. Nomally, the KAN peak signal is out of ange and should not be used as a efeence gene fo nomalization of expeimental genes. If it is desied to use KAN as a efeence gene fo nomalization, the amount of KAN RNA needs to be educed so that the peak height of KAN is simila to that of the median-expesses in the multiplex. 4.4 expess Analysis Question Can I impot data fom two o moe plates into expess Analyze? Yes. Answe See "Impoting Two o Moe Plates into expess Analyze" on page 39 fo instuctions. 34 GenomeLab GeXP

39 Questions & Answes expess Analysis Can I pint o save the Heat Map o othe visual fomats geneated fom my data in expess Map? Ae thee any thid-paty softwae pogams that I can use with GeXP-geneated data fo gene expession analysis? No. Though it is not possible to save, expot o pint gaphs geneated in expess Map, use the Pint Sceen keystoke to captue the sceen image. Paste the sceen captue into Micosoft Wod, Excel o Paint, then save and pint this new file. Yes. IMPORTANT Do not install thid-paty softwae on the expess Pofile contolle fo futhe analysis of gene expession data. The following analysis can be pefomed on GeXP data, using Micosoft Excel: multiple efeence gene nomalization calculation of fold-change statistical analysis Additional softwae pogams fo use with GeXP-geneated data ae GeNom, DecisionSite, PatekGS and Pism. Visit the websites below fo infomation on these softwae pogams. Thid-paty softwae infomation: Toubleshooting Guide 35

40 Questions & Answes expess Analysis 36 GenomeLab GeXP

41 Appendix A Toubleshooting How Tos AAppendix A A.1 Toubleshooting How Tos The following sections ae fo use with completing the toubleshooting pocess as peviously efeenced. Toubleshooting Multiplex Design with Vebose Mode 1. To activate Vebose Mode when designing a multiplex, select the Multiplex genes and/o pimes. 2. Set the Multiplex paametes. 3. Select the option fo Vebose Mode, then click Execute. 4. Identify the unincopoated genes fom the geneated data. 5. Evaluate the accession numbe(s) of the unincopoated gene(s) fo the pope fomat. Accession numbes have necessay chaactes, including undescoes, all of which must be included fo the numbe to be associated with a paticula gene. 6. Evaluate the gene sequence(s) of the unincopoated gene(s) fo the pope fomat, using the FASTA function. IMPORTANT Use only gene sequences that contain A, C, G, T and N. 7. Ente the popely fomatted gene o accession numbe to the multiplex design and click Execute. Repaiing a Coupt TDF File 1. Bowse to locate the multiplex TDF file. 2. Open the TDF file using MS Excel. 3. Veify that all of the multiplex data, including pime names, gene display name and accession numbe ae pesent. 4. In cell A2, change the multiplex name to a unique name. 5. Save the edited TDF file as a text file with a new document name. 6. Open the edited TDF file with Wodpad. 7. Click Edit Replace and eplace all quote maks (") with nothing. 8. Save the file. NOTE Choose a TDF file name that matches the multiplex name (in cell A2) to help keep tack of the diffeent multiplex vesions. 9. Impot the edited TDF file into the database and test this new file fo analysis. Toubleshooting Guide 37

42 Appendix A Toubleshooting How Tos Ceating a Multiplex without an Intenet Connection 1. Log in as Administato. 2. Need steps to get to Manage Genes. 3. Go to the Manage Genes option and pefom the following: a. Copy and paste gene name onto the Display Name field. b. Copy and paste the gene name onto the Accession field. NOTE The Display Name and Accession field must be the same. IMPORTANT Do not use spaces in eithe the Display Name o the Accession field. c. Copy and paste the FASTA desciption onto the FASTA field. d. Copy and paste the gene sequence onto the Sequence field. 4. Click Save. 5. Repeat steps a to d fo all the genes (up to 30 genes) of inteest. 6. Logout as an Administato. 7. Login as a Use. 8. Open the expess Designe. 9. Copy and paste all the accession numbes of the genes of inteest (up to 30 genes) into the Accession Numbes field. 10. Name the new multiplex. 11. Adjust the settings o keep the settings default. 12. Highlight Kan in Refeence Pimes field. 13. Click Execute. A page displaying the designed multiplex will be etuned. If the design is successful, click Save to save the multiplex to the database. If the design is not successful, un Vebose Mode. See "Toubleshooting Multiplex Design with Vebose Mode" on page 37. This multiplex TDF file can be expoted. The pime sequences can be viewed by opening the TDF file with Micosoft Excel. Designing Pimes to Detect Altenative Tanscipts NOTE Even if altenate tanscipts have diffeent accession numbes, the use of these diffeent accession numbes will not necessaily geneate unique amplicons in expess Designe. Thee ae two methods that can be used to geneate unique amplicons: Spanning an Exon To detect the altenative splicing of exon2, design the fowad pime on exon1 and evese pime on exon3. If exon2 is not too big (< 200 nt), the same pai of pimes will detect both vaiants (exon2-included and -excluded), which esults in two distinct fagment sizes on GeXP with the same pime set. NOTE This method will not wok fo the exon2-included fagment if the exon2 is too lage. If exon2 is too big, a evese pime on exon2 can be designed to detect exon2-included vaiant using the same fowad pime. 38 GenomeLab GeXP

43 Appendix A Toubleshooting How Tos Bidging an exon-exon Junction To detect an altenative 5' splice site (exon4 o exon5 spliced to exon6), design unique fowad exon junction bidging pimes. To detect the exon4-exon6 tanscipt, design a fowad pime bidging the exon4-exon6 junction. Make the same type of unique fowad bidging pime fo exon5-exon6 junction. The bidging pime should have 6 to 10 bases paied with the 5' end of distal exon (exon6). The evese pime fo both vaiants can be the same pime diected to the distal exon (exon6). The bidge pime is pefeably the fowad pime because in GeXP chemisty, PCR has highe stingency conditions than RT. Thus, chances fo mispiming with the fowad pimes containing simila sequence ae educed. NOTE If two moe amplicons shae a fowad o evese pime, ode the quantity of that pime as if it wee unique to each amplicon. Modifying a Peviously Designed Multiplex Befoe adding a new set of pimes to a peviously designed multiplex, fist design the pime set with the Pime Design function of expess Designe o ente it as a Refeence Pime though the Administato module, using the Manage Pimes function. 1. Log into expess Pofile and click expess Designe. 2. Click on the Load Multiplex dop-down list, then select the desied multiplex name. The pimes contained in the loaded multiplex become highlighted in the Pimes list field. 3. Use the Ctl+Click function to select the new pime name to be included with the existing multiplex. NOTE To emove a pime fom the multiplex, Ctl+Click on the pime name to clea it fom the list. Multiple pimes can be added o excluded. 4. Make any othe changes to the multiplex as needed. 5. Ente a unique multiplex name in the Multiplex Name field. 6. Name the new settings in the Save Setting field and click Save Setting icon to save the changes in paametes. 7. Click the Execute to ceate the new multiplex. To save the new multiplex to the database, click on the Save to Database. To expot the new multiplex TDF file, click Expot and then bowse and save the file to the desied location. Impoting Two o Moe Plates into expess Analyze Thee ae two methods of impoting multiple plates: combining the data o Impoting multiple CSV files. In eithe case, each of the plates has to be set up (wells identified), peaks binned and saved sepaately befoe poceeding with Nomalization. Combining the Data Befoe impoting the data to expess Analyze, combine all of the data, fom multiple plates, into one study in Fagment Analysis and then use the Tansfe Fagments fo GeXP option which ceates a combined CSV file. When this is impoted into a New Analysis, expess Analyze ecognizes the sepaate plates. Toubleshooting Guide 39

44 Appendix A Toubleshooting How Tos 1. Ceate a new study in Fagment Analysis and combine all of the analyzed data in that study. 2. Tansfe the fagments fo GeXP. This ceates a combined CSV file. 3. Ceate a New Analysis in expess Analyze. 4. Bowse to select the combined CSV file. 5. Impot all of the plates at one time. 6. Continue with Step 1 of Plate Setup. Impoting Multiple CSV Files 1. Ceate a New Analysis in expess Analyze. 2. Bowse to select the CSV file fo each plate. 3. Impot all of the plates at one time. Plate Setup 1. Click on the Plate Setup tab and open the fist plate. 2. Poceed with Plate Setup and Binning, then save the data fo that plate. 3. Open the next plate and epeat steps 1 and 2. IMPORTANT Thee is no visual indicato in the Plate Setup tab to show that anothe plate is waiting to be set up o how many plates ae pat of the analysis. Make sue to keep tack of this infomation to get the desied analysis by impoting multiple plates. When steps 1 and 2 have been completed fo all plates, you can poceed with nomalization. 40 GenomeLab GeXP

45 Appendix B Refeence Figues BAppendix B B.1 Refeence Figues The following images ae fo use with the "Refeence Tables" on page 17 and "Questions & Answes" on page 31 of this guide. Making Pimes Globally Available Log in as an Administato and select the Manage Pimes option. Select the Set as Refeence Pime option fo a pime. Figue B.1 Manage Pimes Sceen - Set As Refeence Pimes Toubleshooting Guide 41

46 Appendix B Refeence Figues Undesigned Peak (UDP) A UDP that co-migates with a designed peak will affect quantitation of the designed peak. Figue B.2 Co-migating Undesigned Peak Repeat Sequences Repeats in the amplicon sequence can cause DNA polymease slippage, which esults in stutte peaks. Figue B.3 Repeat Sequence and esulting Stutte Peaks 42 GenomeLab GeXP

47 Appendix B Refeence Figues Ove-ange Signal in an Unbalanced Pofile A balanced multiplex pofile is achieved by attenuating the evese pime concentation of the high expesses. See Figue B.8 on page 45 fo a Balanced GeXP Pofile. Figue B.4 Unbalanced GeXP Pofile - Ove-ange Ove-ange Signal in Balanced Pofile Any signal >120,000 RFU in analyzed data lies outside the linea ange of detection, with standad GeXP data analysis. Pe-dilute the PCR poduct to bing all fagments within the linea ange. Figue B.5 Balanced GeXP Pofile - Ove-ange Toubleshooting Guide 43

48 Appendix B Refeence Figues Genomic DNA Contamination Genomic DNA contamination of RNA samples leads to the poduction of gene peaks in the RT minus eaction. Figue B.6 Gene Peaks in RT minus eaction Signal Dop-Off Smalle fagments have a much highe signal than lage fagments, due to themal cycling tempeatue fluctuation. Figue B.7 Electopheogam with Signal Dop-off 44 GenomeLab GeXP

49 Appendix B Refeence Figues Dye-Channel Pull-up If the D4-dye signal is too stong, a small geen D3-dye peak may be obseved undeneath the ove-anged peak. Figue B.8 D3-dye Channel Pull-up Toubleshooting Guide 45

50 Appendix B Refeence Figues Unesolved Potein-Nucleic Acid Complex A non-specific potein-nucleic acid complex will consistently migate in a paticula size egion, specific to the multiplex. Figue B.9 Unesolved Fagment 46 GenomeLab GeXP

51 Appendix B Refeence Figues Exclusion Filte The pupose of an exclusion filte is to exclude any data that does not meet the citeia of the study, and to include the esults that do meet the citeia. The figue below demonstates that valid peaks ae not called when the wong exclusion filte is applied. Fo moe infomation, see Chapte 7, Applying Exclusion Filtes to the Fagments List in the GenomeLab Genetic Analysis System Use s Guide (A29142). Figue B.10 Impope Exclusion Filte Applied Figue B.11 Sample GeXP Exclusion Filtes - Basic & Extensive Toubleshooting Guide 47

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