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480 System Technical Manual #TM290

1. Promega XI D Manual Baseline Adjustments The proper baseline region is important for optimal analysis of Plexor System data Baseline regions are automatically determined during import of data into the Plexor Analysis Software The baseline region is set in a flat region of the amplification curve before the beginning of the downward inflection that indicates product accumulation In some instances manual adjustment may provide optimal representation of the data This may include samples with excessive noise bleedthrough or early C values or situations where the real time instrument shows early signal fluctuation 1 Select the PCR Curves tab 2 Select the Display and Manually Adjust Baselines icon lex 3 Select the samples to be adjusted using the well selector Note The baseline region can be adjusted for individual samples or groups of samples by selecting or dragging the lower and upper limits The shading in the baseline region will be gray if the selected samples do not share a common baseline region Figure 19 For multiplex assays the baseline is set independently for each dye PER cere Semple oa o Rn Baseline FAM Target 1 208 Tagara Lower limit region 5510TA Upper limit Figure 19 An amplification window showing the baseline region and baseline upper and lower limits Adjust the upper limit of the baseline region for each sample so the upper limit is approximately 5 c
2. 23 28 DBermebt a Natal es eed ED a A OEA nd NM ER 23 WEE cocos m 27 VIL Data Export from the Roche LightCycler Data Analysis Software 28 VIII Data Import into the Plexor Analysis Software ses 99 IX Data Analysis with the Plexor Analysis Software se 36 Ao mne Dem NO oneee 36 b Adjusting the Expected Target Melt Temperate nosa 38 C Adjusting the Y Axes of the Amplification and Tiermal Melt Curves Omnia Luise leise rte Snarare aane reiii S aara EHI Roots et AEA 40 D Adjusting the Baseline Region and Melt e E E je ute 1121 A EU INED yy nr EE eU 40 BE Generating a Standard Curve Opto iia siitiia sisson ssscccctscumemanaamaneeostalieareaveoueret 41 EB O A E E 4 oe ond Pining ME Anys SUDO eceieteoseeeeni MUCH ORDER UL rese rtota uses dd 46 AX POU CS IN e oou EEUU 46 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 n Page 1 C NEP Dun e ae oan nee 53 i A Plexor Analysis Software Operating System Compatibility 53 eecoooocooooooooooooooooooooooo Pro B Plexor Analysis Software Installation eene 53 mega C AT AEE E E EEEE 22 D Manual Baseline AGUS er neue oe eh tk ma RMMLA R 56 Bi 0 DRITTER UNUM A MEINER UNE Mm rTerver a 97 B Amplification Bfi
3. button in the lower window With the 60 C target temperature selected change the acquisition mode to Single OO 00305 wou 00 35 6458TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Printed in USA Page 6 5 07 14 Next to Program Name select the button to add a program Name C the third program of this experiment Melt or a similar title In the Analysis Mode pull down menu choose Color Compensation Promega 15 With the Melt program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window With the 95 C target temperature selected change the acquisition mode to Continuous and change Acquisitions per C to 2 5 lada ihh i vrais T re r Acow DIES Sec Ta E 7 jis ys 4 m ei rene onomos fet 2 3 el Wone 00 00 30 BE js deine z ES o cru om uiu E 6459TA 16 Next to Program Name select the button to add a program Name the fourth program of this experiment cooling or a similar title In the Analysis Mode pull down menu choose None 6460TA x no no 30 gs 17 With the cooling program name selected change Program Temperature Targets to the values indicat
4. icon to add a program In the Program Name section name the first program of this Pre experiment Plexor first step genotyping or a similar title In the m g Analysis Mode pull down menu select None 6 With the Plexor first step genotyping program name selected change Program Temperature Targets to the values indicated below A ST Propran 7 Wrieror 2 firot step genotyping zi Quantification Belt ing Curves poen Rt Joo 00 3 s o 6477 TA 7 Next to Program Name select the button to add a program Name the second program of this experiment PCR or a similar title Enter the number of amplification cycles In the Analysis Mode pull down menu select Quantification 8 With the PCR program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window With the 60 C target temperature selected change the acquisition mode to Single iprggam Name 58 7 fffriexce irat acep genotyping c pum peR 80 0 0 0 0 0 0 0 0 0 0 wan iticntion EX tele ji eiting Curves 6478TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 oe Page 25 C 9 Next to Program Name select the button to add a program N
5. Analyses pull down menu From the Create new analysis list select Abs Quant Fit Points Tm Calling 6481TA 4 Inthe Create new analysis pop up window confirm that PCR is selected in the Program pull down menu Selecting All Samples in the Subset pull down menu will result in analysis of the entire plate Alternatively if the experiment contains previously defined subsets of samples a specific subset can be chosen for analysis Select OK to proceed Create new analysis Analysis Type f Quant Fit Points Subset s Samples Program FcR Nam fis Quant Fit Points for All Sample PEPERISEEPRISISPIE 6482TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 28 Spur Amplification curves are displayed in the chart For multiplex data i e more than one dye a color compensation file must be applied Select the Color Comp button and choose In Database From the Available Color Compensations list select the appropriate color compensation file and select OK In the Color Compensation Channels box choose the channels to compensate and select OK a Compensation Channels X gc 1 do EG 6483TA The Filter Comb button indicates the color channel being displayed To view each color chann
6. Increasing fluorescence over time Excessive template was added to the reactions Dilute the template and re amplify The baseline region was set in a region with significant fluorescence fluctuation The baseline within the baseline region should be flat Manually adjust the baseline region Section XI D The baseline region was set too close to the signal change Manually adjust the baseline region Section XI D Two or more distinct melt curvesin For the Plexor qRT PCR Systems both RNA and DNA the melt curves window templates can be amplified Treat the RNA template with DNase to eliminate contaminating genomic DNA Poor primer specificity Design new primers with higher specificity to the target To verify primer specificity perform a BLAST search with the primer sequence The primer should not exhibit regions of identity with other sequences Optimize the annealing temperature Increase the annealing temperature by increments of 2 C to reduce the synthesis of primer dimers or nonspecific amplification products Pseudogenes or polymorphic genes may exist Design new primers to avoid regions of identity between gene family members Assemble the reactions on ice to minimize the synthesis of primer dimers or nonspecific amplification product Reduce the number of amplification cycles to minimize the synthesis of primer dimers or nonspecific product Check for signal bleedthrough Calibrate the instrument as instructed by the manufacture
7. The scale of the Y axis of the amplification curve was affected by other reactions on the plate A high fluorescent signal for one or more reactions can cause the scale of the Y axis of the amplification curve to be too high to see changes in some data Adjust the scale of the Y axis to accommodate samples with smaller changes in fluorescence See Section IX C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 49 Promega X Troubleshooting continued Symptoms Nonlinear standard curve low R values Causes and Comments An amplification inhibitor was present in the standard reference template Determine whether the template contains inhibitors by adding the DNA template to the positive control reaction a significant increase in the C value or no amplification of the positive control in the presence of the DNA template indicates the presence of inhibitors Repeat purification of the standard reference template used to generate the standard curve Calibrate your pipettes to minimize variability in pipetting Small volumes are difficult to pipet accurately Do not pipet volumes 1yl dilute the template so larger volumes are pipetted Viscous samples e g high molecular weight genomic DNA are difficult to pipet accurately Dilute the DNA template Shear high molecular weight D
8. 1e1 1e2 1e3 etc Standard reference templates with the same concentration may be assigned simultaneously by highlighting multiple wells The software does not accept commas in the concentration assignments Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 37 4 A row or column of contiguous wells within a dilution series of standard reference templates may be simultaneously assigned as standards using the Assign Dilution Series function by highlighting multiple wells Figure 7 You must enter the initial concentration of the series the dilution factor and whether the series is increasing or decreasing 5 Colors can be assigned to samples to provide distinction to the displayed data Select the samples then select color assignment to apply a color to the selected sample s Assigning or changing a color does not alter the information associated with a sample For example the concentrations of standard samples Will be retained if the display color associated with those samples is changed Assign Dilution Series Horizontal Series Starting Concentration fi Dilution Factor fio Increasing Decreasing Apply Cancel 5049TA Figure 7 The Assign Dilution Series screen IX B Adjusting the Expected Target Melt Temperature Set the expected target m
9. My Computer Traceable ag Wii re 9 ovens Lisci Sytem Admin ms II T H OS Tools button Select Detection Formats then choose New and select the appropriate instrument A new detection format will be created E User Access l Current Password Users and Groups System Settings Beport Settings B Database information View logged in users Update query engine Clean up database Instruments 6454TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 Partt TM290 Page 4 WWW promega com Printed in USA 5 07 4 Select the appropriate excitation and emission filter combinations for the e dyes in your multiplex using the Filter Combinations Selection window Set the max integration time to 0 25 for all four color channels Promega B User Access Curtent Peseword Users and Groups System Settings Report Settings G Database infor ation View logged in users Update query eagine Clean up database I 6455TA 5 Select Close to save the new detection format into the database and return to the startup screen 6 Toaccess this newly created detection format the LightCycler 480 software must be closed then reloaded 7 After closing and reloading the software select New Experiment in the startup screen 8 Cho
10. Plaase fill in ihe details below regarding your run Run Details Assay Name Cancel Back Next gt 6491TA Figure 2 The Run Info screen Select Next Use the File Import screen Figure 3 to specify the data files exported from the instrument in Section VII Use Browse to locate the appropriate exported amplification and dissociation data files When analyzing data with the Plexor Analysis Software be sure to choose the amplification curve file and melt curve file generated using the same data The file names assigned when exporting data must be descriptive so that the appropriate files can be easily identified and imported into the Plexor Analysis Software Note Advanced Options can be used to create templates for routine plate setups and analysis conditions See Section XI C for details concerning these advanced options and an explanation of the default analysis settings Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 34 Printed in USA 5 07 File Import b To import your LightCycler480 run use the file dialogs below to specify the files for each dye used for amplification and melt as labeled below Finish 6492TA Figure 3 The File Import Screen 10 Select Finish to complete the data import and to open Analysis
11. The software does not accept commas in the concentration assignments Standard samples are displayed as circles in the well selector and standard curve graphs They are labeled standard in reports gis Create Dilution Series shortcut f The Create Dilution Series function creates a full dilution series within a row or column of capillaries Select the capillaries that contain a dilution series of the standard then select Create Dilution Series You must enter the initial concentration of the series the dilution factor and whether the series is increasing or decreasing Figure 14 Concentrations may be entered in standard format 0 01 0 1 1 10 100 1000 etc or scientific format 1e 2 1e 1 1e0 1e1 1e2 1e3 etc The software does not accept commas in the concentration assignments All selected capillaries will be assigned the sample type standard with the appropriate concentration This function can only be performed with standards within the same row or column Using this function produces the same result as selecting each capillary in the series individually and assigning it the sample type standard with the appropriate concentration Only samples that have been assigned the sample type standard will be used to generate the best fit line in standard curves Standard samples are displayed as circles A row or column of capillaries within a dilution series of standards may be assigned as standards simultaneou
12. This can be avoided by exporting the samples for each assay separately Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Page 32 Printed in USA 5 07 VIII Data Import into the Plexor Analysis Software e The Plexor Analysis Software Cat A4071 is available for download at P www promega com plexorresources It is also available free of charge on CD ROM mega by request Software installation instructions are given in Section XI B When exporting data for use with the Plexor Analysis Software be sure to assign descriptive names to the files so that related amplification curve and melt curve files e g files generated using the same data can be easily identified during data import 1 To launch the Plexor Analysis Software go to the Start menu and select Programs then Plexor select Analysis Desktop Note A shortcut can be placed on the desktop by right clicking on Analysis Desktop selecting Copy then right clicking on the Windows desktop and selecting Paste Shortcut R 2 Inthe File menu select Import New Run or select the icon 3 Optional Enter an assay name in the Assay Setup screen Figure 1 This screen is used to enter general information about the type and format of the data that will be used for each assay 4 Select Roche LightCycler 480 a
13. XLE Icon Definitions Assign Color shortcut q The Assign Color function allows you to select a color in which a sample is displayed This color selection is associated with those samples in the amplification and melt curves well selector and any reports Select one or more capillaries using the well selector then select this button to choose the desired color for the selected samples These colors are not transferred to printed copies or exported reports Sample color does not change the analysis of a sample in any way ee Assign Unknown shortcut w The Assign Unknown function allows you to assign the sample type unknown to all selected samples Select one or more capillaries using the well selector then select this button to assign the sample type unknown Unknown samples are displayed as open squares in the well selector They are labeled unknown in reports When included in a standard curve the concentrations of unknown samples will be calculated and reported e Assign NTC shortcut e The Assign NTC function allows you to assign the sample type no template control to all selected samples Select one or more capillaries using the well selector then select this button to assign the sample type no template control No template control reactions are displayed as diamonds in the well selector They are labeled as no template control in reports When included in a standard curve th
14. to return to the Experiment window 5 Load the reaction plate into the LightCycler 480 and immediately press Start Run to begin thermal cycling Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Printed in USA Page 22 UM VI Instrument Setup and Thermal Cycling for Genotyping SNP Assays e y These instructions describe instrument setup and thermal cycling conditions for genotyping assays using the Plexor qPCR System This cycling program is specific for genotyping primers designed using the Plexor Primer Design Software Thermal cycling programs described in this manual are optimized to work with primers designed using the Plexor Primer Design Software The Plexor Primer Design Software can be accessed at www promega com plexorresources Promega VI A Thermal Cycling Program The thermal cycling program is shown in Table 5 The cycling program will include one cycle with an annealing temperature of 50 C followed by 40 cycles with an annealing temperature of 60 C The first round of PCR is performed at the lower annealing temperature additional rounds of PCR are performed at the higher annealing temperature to increase the specificity of amplification Table 5 Thermal Cycling Profile for Genotyping Assays Step Temperature Time Number of Cycles Initial Denaturation 2 minutes 1 cycl
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16. Redesign your primers so the melting temperatures are 60 C We strongly encourage using the Plexor Primer Design Software Viscous samples e g high molecular weight genomic DNA are difficult to pipet accurately Dilute the DNA template Shear high molecular weight DNA by vortexing or pipetting Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Printed in USA Page 48 SP X Troubleshooting continued e y Symptoms Causes and Comments Variability in signal among The baseline region was not set correctly The baseline should Pro mega replicate samples continued be flat The baseline region can be adjusted manually for each well to account for sample to sample variation Section XI D Be sure the reaction plates are properly sealed to avoid evaporation Fluorescence decrease observed in Nonspecific product can accumulate at higher cycle number in the no template control reactions with targets present at low copy numbers Assemble the reactions on ice to reduce the accumulation of nonspecific amplification products Decrease the cycle number to reduce the accumulation of nonspecific amplification products Design new primers using the Plexor Primer Design Software Reactions were contaminated with target DNA or RNA Clean workstations and pipettes with a mild bleach solution before and after use Use
17. melt curve has the lowest i e the most negative d RFU dT value The default melt threshold d RFU dT percentage is preset at 25 0 and can be set between 0 0 and 100 0 This value is used by the software to calculate the melt threshold value The T threshold value is defined as a percent of the d RFU dT value for the sample with the lowest d RFU dT value in the data set The melt threshold value is recalculated when a standard curve is generated The melt threshold value can be manually adjusted by clicking and dragging the horizontal melt threshold line Expected Target Melt Temperature The expected target melt temperature is the melt temperature of the correct PCR product The expected target melt temperature must be between 65 C and 95 C The default expected target melt temperature is 90 C Target T Upper Bound The target T upper bound is the number of degrees Celsius above the expected target melt temperature at which a sample T is considered to be suspect The default target T upper bound is 1 C Target T Lower Bound The target Tm lower bound is the number of degrees Celsius below the expected target melt temperature at which a sample T is considered to be suspect The default target T lower bound is 1 C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 am Page 55
18. new reagents and solutions Take precautions to prevent contamination see the Plexor qPCR System Technical Manual TM262 the Plexor One Step qRT PCR System Technical Manual TM263 or the Plexor Two Step qRT PCR System Technical Manual TM264 An improperly calibrated instrument can lead to erratic fluorescence readings Calibrate the instrument as instructed in Section III or per manufacturer instructions Vertical fluorescence spikes or Consult the instrument manufacturer s user s manual for significant noise in the information about potential instrument problems that can amplification curve cause spikes or noise No amplification or poor amplification for the entire run Poor amplification can lead to improper data scaling making the fluorescence measurements appear erratic See possible causes and comments for Flat amplification curve in the amplification curves window no apparent amplification above Instrument was improperly calibrated Calibrate instrument as instructed in Section III or per manufacturer instructions Small signal change in No amplification or poor amplification See causes and amplification curve and melt comments for Flat amplification curve in the amplification curves curves window no apparent amplification above Incorrect filter was selected Verify the presence of the appropriate filter Primer concentration was incorrect Verify primer concentration by measuring the absorbance at 260nm
19. the Program Name section name the first program of this Pre experiment Denature or a similar title In the Analysis Mode pull m g down menu select None 6 With the Denature program name selected change Program Temperature Targets to the values indicated below 6468TA 7 Next to Program Name select the button to add a program Name the second program of this experiment PCR or a similar title Enter the number of amplification cycles In the Analysis Mode pull down menu select Quantification 8 With the PCR program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window With the 60 C target temperature selected change the acquisition mode to Single p ai i p Sd rims i Es 2 Rum ification Melting Curves i Em E te 55 6469TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 oe Page 15 10 T 12 Next to Program Name select the button to add a program Name the third program of this experiment Melt or a similar title In the Analysis Mode pull down menu select Melting Curves With the Melt program name selected change Program Temperature Targets to the values indicated below To
20. 3TA DTe Torezheolz Figure 10 The Standard Curves tab A standard curve with the log concentration on the Y axis and the cycle threshold on the X axis i Standard Curve 1 Well Sampie Mm Standent 0C 780 784 opo ve J 4 tndaM UO 279 794 UE ves y 3 27x 34 34 Standart 00 270 TRS IOGT Ves R 0 993 Sampie 1 363 792 20602 vw Sampie 2 278 702 95EO0 Yes Sample 274 702 139802 Ver Standard 526 3270 788 19280 Yes Standart 124 Mh TRE 260 Ves Standard 526 Oo SE 12580 Ves Sample 4 3210 792 1000 Ver Sampie s 2310 702 0760 vu Sample 310 792 toet Ver Stadant 2 5 BO RR 2580 Ys Standa 2 5 320 702 2660 Ye Standa 25 3229 792 2460 Ye Sample 343 792 10600 ver 3 Sample 0 250 792 6400 Ye 1 Sample 4 792 47EOD ves Standa 054 36 702 54EO Yes Sanda 05S 3553 702 S4EOt ves i Standa 0Sa 354 792 54EO Yer e Sample 10 4 795 SIEN Ver a Sample 1 377 795 02602 Ye Sample t2 303 7065 35602 Ye t Standen 0 8 5775 92 SSE Ver Standar 0 18 mo 706 18EO ve Standard 0 50 3 795 11600 Ye Sample 13 201 7S 3 3280 Ver Sample 14 6 7S 22002 Ver Sample 15 25 705 25602 Yn Standart 0 06 32 MUS O0EN Y Stand ant 0 06 390 705 006 Y 1 Q3 NIC dors not have a valid CI value 3 Stands 006 586 795 625622 ve R H2 NTC does not have a valid Ct value 4 Sample 16 366 792 28602 Yes OHI NTC dows not have a valid Cl value S Sample 17 260 702 34602 vu 6 Sample t 400 U2 22802 Ver Standaet O07 ani
21. 4 4330 Fax 608 277 2516 www promega com Partt TM290 Page 52 Printed in USA 5 07 XI Appendix C XI A Plexor Analysis Software Operating System Compatibility The Plexor Analysis Software is compatible with the following operating systems Windows 98 Windows NT 4 Windows ME Windows XP and Windows 2000 Other operating systems are not supported The Plexor Analysis Software is not compatible with Macintosh computers Be sure that the display settings for the computer are set to 32 bit color rather than 16 bit color when using the Plexor Analysis Software XI B Plexor Analysis Software Installation The Plexor Analysis Software and installation instructions are available for download at www promega com plexorresources The software is also available free of charge on CD ROM by request Consult the Promega Web site to verify that you are installing the most recent version of the software Following installation the program can be accessed in the Start menu Programs VPlexorV Analysis Desktop Instructions for Installing the Plexor Analysis Software from CD ROM 1 Insert the CD ROM into the CD ROM drive 2 Double click the Plexor exe installer icon on the CD ROM and follow the on screen instructions to install the software Note Installation of the software may take several minutes There is a pause where the computer may appear to be inactive between the launch of the installer and th
22. 711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 oe Page 17 Instrument Setup and Thermal Cycling for One Step qRT PCR These instructions describe instrument setup and thermal cycling conditions for cDNA quantitation using the Plexor One Step qRT PCR System The thermal cycling program includes the initial incubation for the reverse transcription Thermal cycling programs described in this manual are optimized to work with primers designed using the Plexor Primer Design Software The Plexor Primer Design Software can be accessed at www promega com plexorresources V A Thermal Cycling Program The thermal cycling program is shown in Table 4 Primers designed using the Plexor Primer Design Software have an annealing temperature of approximately 60 C Table 4 One Step qRT PCR Thermal Cycling Program Step Reverse Transcription Initial Denaturation and Inactivation of the ImProm II M Reverse Transcriptase Denaturation Annealing and Extension Denaturation Melt Temperature Curve Instrument Cool Temperature Time 45 C 5 minutes 95 C 2 minutes Y 5 seconds 35 seconds 95 C 5 seconds 50 C to 95 C 2 5 acquisitions per C 50 C 30 seconds Number of Cycles 1 cycle 1 cycle 40 cycles 1 cycle 1 cycle 1 The length of incubation for the reverse transcription reaction can be in
23. 792 20602 Ve Standa OOI Wt M2 ZOEN Ver 03 St dac002 2902 792 25602 Ver PII 5054TA Figure 11 The Standard Curve tab A standard curve with the cycle threshold on the Y axis and the log of the concentration on the X axis Note Samples that do not cross the amplification threshold such as the no template controls are listed in the graph as not having a valid C value Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 ne Page 43 IX F Reports The Plexor Analysis Software includes five report options Sample Details Thresholds Baseline Regions Run Info and Import Files which are included as subtabs in the Reports tab To view these report options select the Reports tab Information is presented in a tabular format that can be copied saved or printed using the provided icons The saved data can be opened using Microsoft Excel For multiplex assays the Plexor Analysis Software reports include information for all of the dye labels Sample Details The sample details report includes well location sample ID dye channel cycle threshold thermal melt temperature concentration if applicable whether the sample has the expected T and the number of melt curves that cross the melt threshold line Figure 12 A sample with a C value of N A has an ampli
24. Desktop Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 oe Page 35 CQ IX Data Analysis with the Plexor Analysis Software A After data import is complete Section VIII the PCR Curves tab of the Analysis Promega Desktop is displayed Figure 4 Tools Tab Selection TT eene enr Faas Taste 8 Jom Tages Amplification Curves Window Melt Curves Window Well Selector E E 5046TA Figure 4 The PCR Curves tab of the analysis desktop The amplification curves window melt curves window and well selector are indicated IX A Sample Definition E Use the Well Selector which is shown in Figure 4 to select and define each well or group of wells Choose one of the icons shown in Figure 5 to define the samples See Notes 1 5 To assign sample names select the Sample IDs tab Figure 6 To enter sample names manually select the well and enter the desired sample name Repeat to enter sample names for other wells To copy names from a MicroSoft Excel spreadsheet highlight the sample names in the spreadsheet and select copy In the Edit menu select Paste Sample IDs from Template or use the control T shortcut The layout of the sample names in the spreadsheet must be the same as the layout of the
25. NA by vortexing or pipetting Adjust the baseline region The baseline region can be manually adjusted for each reaction See Section XI D Some variation is normal Perform duplicate or triplicate reactions for the standard curve to minimize the effect of this variation There will be statistical variation in the amount of template in a reaction with targets present at low copy number Perform duplicate or triplicate reactions for the standard curve An error was made during dilution of the standard reference template Verify all calculations and repeat dilution of the standard reference template Do not pipet volumes 1l Incorrect concentration values were entered in the Plexor Analysis Software Verify the concentrations for all samples used to generate the standard curve Reactions were contaminated with target DNA or RNA Clean workstations and pipettes with a mild bleach solution before and after use Use new reagents and solutions Take precautions to prevent contamination see the Plexor qPCR System Technical Manual TM262 the Plexor One Step qRT PCR System Technical Manual TM263 or the Plexor Two Step qRT PCR System Technical Manual TM264 Carefully seal the reaction plate to avoid evaporation Aberrant fluorescence can be caused by contamination fingerprints etc Do not write on the surface of the optical adhesive plate covers Use caution when handling optical adhesive plate covers Wear gloves Promega Corp
26. Notes 1 and 2 View the concentrations for all samples including the unknown samples in the table next to the standard curve graph Figures 10 and 11 The calculated concentrations can also be viewed in the sample details report Section IX F Repeat Step 1 with any other desired set of standards and samples Notes t A second standard curve can be created using a different set of samples Repeat Step 1 using the new set of standard samples Multiple standard curves may be created within the same data set if none of the samples and standards are shared If you attempt to generate an additional standard curve using samples that are used in the current standard curve the alert box Confirm Standard Curve Replace will appear The currently assigned standard curve will be overwritten with the new standard curve if OK is selected An existing standard curve can be changed or additional unknowns added To do so delete the existing standard curve Go to the Standard Curves tab then select Remove Standard Curve The Remove Standard Curve button is only active in the Standard Curves tab NS Remove Standard Curve Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 42 Printed in USA 5 07 Banded Cursos 1 Wail Sarpila HD i Tm Cura TE Tm A Sada dC 20 784 BEDT Yas mega 1 Stu
27. See Section X for more information about possible causes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA 5 07 Partt TM290 Page 39 The target melt temperature range can be adjusted manually Move the mouse so that the arrow is over the upper or lower limit and drag the limit to the desired temperature Alternatively upper and lower limits can be adjusted by double clicking on the appropriate lines and entering an exact value in the pop up window that appears The melt threshold is the level of signal that must be reached for the Plexor Analysis Software to call the melt results Target Tm indicators are included in the table to the right of the amplification and melt curve windows A Yes or No in the Tm column indicates whether a sample Tn is within the expected target melt temperature range A No Call in this column indicates the melt curve displays the correct expected target melt temperature but there is insufficient amplification product to cause the amplification curve to cross the melt threshold The Tm is the number of peaks that cross the melt threshold line More than one peak indicates heterogeneous amplification products This may be due to nonspecific amplification secondary structure or a polymorphic target See Section X for more information about possible causes C
28. Technical Manual Plexor Systems Instrument Setup and Data Analysis for the Roche LightCycler 480 System INSIRUCTIONS FOR USE OF PRODUCTS A4011 A4021 A4031 A4041 A4051 AND A4061 PRINTED IN USA 5 07 Part TM290 Plexor Systems Instrument Setup O and Data Analysis for the Roche omega LightCycler 480 System All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this Technical Manual I NSN HO eneen EA EE E 2 I Plate Preparation and A mip litt cath Om ssisssaccsssczsvesvsnsncexvenstuveassnsnveaseasascosnsseacessopanssasonensenvanseanan 2 Ill Generating Color Compensation FI es un ipie EE Ett teceseinns 3 A Thermal Cycling Program for Color Compensation se 3 b Color Compensation cample EODD Oeo e onis ce cerneret mme ce HERR 8 C Caor Con pena or Reden on DeL Duaceese coute to ioter eei RUE ue 10 D Creating a Color Compensation Analysis File sss 11 IV Instrument Setup and Thermal Cycling for qPCR and Two Step qRT PCR 13 fe Mammy Tero nec Gye Vi akon PEDOEGD anotar teet po ed DD posce ERAI oie e DUE 13 MEE desi 17 V Instrument Setup and Thermal Cycling for One Step qRT PCR 18 MM Disc nd uiti T To 00 Ln 18 ES vuelos D T IQ 22 VI Instrument Setup and Thermal Cycling for Genotyping SNP Assays
29. The default threshold is 10 standard deviations but can be changed in the Analysis Template or recalculated at any time by using Set Amp Threshold from Selected Samples option in the Edit menu See Section IX D Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 54 5 07 BENE Analysis Defaults Setthe default parameters for analyzing the targets in this assay Promega DPUR amp EF C 9C I E EI TTL Deo eee FAM Target 1 JOE Taret 2 a 1 i AeL i ee bod rares Default Amp Threshold RFU Baseline Noise Standard Daviatisns o HN Default Melt Threshold CARFUYAT Percentage 0 0 100 0 D50 Expected Target Melt Temperature Tm 95 0 96 0 C ooo Tames E 5061TA Figure 18 The Analysis Defaults tab Default Melt Threshold d RFU dT Percentage The melt curve allows you to distinguish amplification products with different sequences and lengths In the absence of nonspecific amplification products the melt curve will have one peak Each sample has a melt curve from which a T can be determined A T value is reported for all melt curves that cross the melt threshold The melt threshold represents the d RFU dT value that is required before a Tn value is reported for a sample A sample s Tn value is calculated as the temperature at which the
30. add new steps select the button in the lower window With the 95 C target temperature selected change the acquisition mode to Continuous and change Acquisitions per C to 2 5 6470TA Next to Program Name select the button to add a program Name the fourth program of this experiment cooling or a similar title In the Analysis Mode pull down menu select None With the cooling program name selected change Program Temperature Targets to the values indicated below 6471TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 16 Printed in USA 5 07 IV B Sample Editing e by 1 Sample information can be entered in the LightCycler 480 software before during or after your experiment is run Alternatively sample Promega names can be entered during the data analysis step using the Plexor Analysis Software 2 To enter sample information using the LightCycler 480 software select Sample Editor 6472TA 3 Under the General tab indicate which are replicate wells Name the samples if desired 4 Select Experiment to return to the Experiment window 5 Load the reaction plate into the LightCycler 480 and immediately press Start Run to begin thermal cycling Promega Corporation 2800 Woods Hollow Road Madison WI 53
31. ame e the third program of this experiment Melt or a similar title In the Promega Analysis Mode pull down menu select Melting Curves 10 With the Melt program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window With the 95 C target temperature selected change the acquisition mode to Continuous and change Acquisitions per C to 2 5 40 lt lQuantification z po greim cuves 7 6479TA 11 Next to Program Name select the button to add a program Name the fourth program of this experiment cooling or a similar title In the Analysis Mode pull down menu select None 12 With the cooling program name selected change Program Temperature Targets to the values indicated below Il exe first exor first step gerotyping 000000000000000000000000000000000000000 6480TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Printed in USA Page 26 UM VLB Sample Editing e by 1 Sample information can be entered in the LightCycler 480 software before during or after your experiment is run Alternatively sample Promega names can be entered during the data analysis step using the Plexor Analysis Software 2 To enter sample information using the LightCycler 480
32. ands 008 390 Fea jti 1 Hi HIC Hs HTC Graph Legend showing Sample T and Target T indicators lt Melt Threshold mg re Bo i i 9051 TA Tampa Cy Figure 8 The expected target melt temperature is displayed in a table to the right of the graph In the melt curves window move the mouse so the arrow is over the expected target melt temperature line and drag it to the desired temperature Alternatively double click on the line and enter the desired temperature See Notes 2 5 Optional The melt threshold may be reset to change the sensitivity in detecting the amplification product See Note 6 The default melt threshold is set at 2576 of the signal change for the sample within the set that has the greatest change in signal In some instances the sample s used to set this threshold may not be typical of the data set To adjust the melt threshold line based on a selected set of samples highlight the desired samples in the well selector In the Edit menu select Set Melt Threshold From Selected Samples Enter the desired percentage of signal change To manually adjust the melt threshold line place the cursor over the threshold line and drag the line to the desired location Alternatively double click on the threshold line and enter the desired threshold value Notes 1 The absence of peaks for the standard reference template or genotyping control sample indicates amplification problems
33. creased to up to 30 minutes Longer incubation times can lead to increased sensitivity but also higher background Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 18 Printed in USA 5 07 1 Open the LightCycler 480 software e be 2 Select New Experiment in the startup screen g liiy b ot iatea release 120085 ME linanimane LESO 1243 Stamdlry no AAP Database My Computer Traceable Mind Overview Luiz System Almin 8m B e 6453TB 3 Select the appropriate detection format from the pull down menu The detection format specifies which fluorescent channels will be read created in Section II A Mano Color Hydrolysis Prose Mei Color Hydrolysis Probe Mano Color HyoPrcbe Huiti Color HytfFrcbe FAH COSB0 CR535 QE TD 6467TA 4 Enter the reaction volume Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 ne Page 19 Tanpet C Acquisitian Made Hold hh mm ss Im T His gone 00 05 00 Eis wn oo o2 00 Saa S chan Valu m Reverse Transcription and deraturaticr iractivation The cycling will consist of four programs Program the in
34. creases tenfold every 3 32 cycles 2332 10 so C values differ by 3 32 for each tenfold dilution A reaction with 100 efficiency will have a slope of 3 32 when the amplification curve is displayed with the C values on the Y axis and log concentration on the X axis When the amplification curve is displayed as C versus log concentration the efficiency may be calculated as 10 slore 1 x 100 1 XI G Reference 1 Bustin S A 2004 A Z of quantitative PCR International University Line La Jolla Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 59 Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process expired on March 29 2005 The purchase of this product conveys to the buyer the limited nonexclusive nontransferable right without the right to resell repackage or further sublicense under U S Published Patent Appln 20020150900 and U S Pat Nos 5 432 272 6 617 106 and 6 140 496 to use the product No other license is granted to the buyer whether expressly by implication by estoppel or otherwise In particular the purchase of this product does not include or carry any right or license to sell this product For information on purchasing a license for other use
35. d to export data from each of the color channels To analyze the melt data select Overview in the Analyses pull down menu Abs Quant znd Derivarive Max iba Quant Fit Points Color Compensation From the Create new analysis list Tm Calling select Tm Calling 6487TA In the Create new analysis pop up window confirm that Melt is selected in the Program pull down menu Selecting All Samples in the Subset pull down menu will result in analysis of the entire plate Alternatively if the experiment contains previously defined subsets of samples a specific subset can be chosen for analysis Select OK to proceed Create new analysis T wne 11 il t A11 Samples hd 3 Tm Calling for All Samples 6488TA Promega Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA 5 07 Part TM290 Page 31 14 19 T6 TA 18 19 20 Melting curves are displayed in the upper chart For multiplex data i e more than one dye a color compensation file must be applied Select the Color Comp button and choose In Database From the Available Color Compensations list choose the appropriate color compensation file and select OK In the Color Compensation Channels box choose the channels to compensate and select OK The Filter Comb bu
36. e Annealing and Extension 35 seconds Denaturation 5 seconds Annealing and 40 cycles Extension 35 seconds Denaturation 95 C 5 seconds 1 cycle Mei Temperate 50 C to 95 C 2 5 acquisitions per C Curve Instrument Cool 50 C 30 seconds 1 cycle Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 23 C 1 Open the LightCycler 480 software 2 2 Select New Experiment in the startup screen Promega B LightCyclerib 400 Software release 1 200625 all gt amame LEAB 1241 Seamdiy no BAP Database My Computer Traceable Mind Overview Urar ymi dudanin ME E Bg ae 6453TB 3 Select the appropriate detection format from the pull down menu The detection format specifies which fluorescent channels will be read created in Section II A Wa Mano Color Hydrolysis Probe Multi Color Hydrolysis Probe Bono Color HybPrcbe Multi Color WybPrcbe FAH COSB0 CRO35 QETO 6467TA 4 Enter the reaction volume Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Printed in USA Page 24 UM 5 The cycling will consist of four programs Program the instrument by e adding the program names in the top window Select the
37. e concentrations of sample in the no template control will be calculated and reported Assign Positive Control shortcut t The Assign Positive Control function allows you to assign the sample type positive control to all selected samples Select one or more capillaries using the well selector then select this button to assign the sample type positive control Positive control samples are displayed as hexagons in the well selector They are labeled positive control in reports When included in a standard curve the concentrations of positive control samples will be calculated and reported Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 57 Assign Standard shortcut r The Assign Standard function allows you to assign the sample type standard to all selected samples Select one or more capillaries using the well selector then select this button to assign the sample type standard Only samples that have been assigned a type of standard will be used to generate the best fit line in standard curves All standard samples must be assigned a concentration by the user when they are defined as a standard Concentrations may be entered in standard format 0 01 0 1 1 10 100 1000 etc or scientific format 1e 2 1e 1 1e0 1e1 1e2 1e3 etc
38. e and generate the new standard curve using the samples you have selected You may generate the original standard curve at any time NS Remove Standard Curve shortcut c The Remove Standard Curve function removes the standard curve on the tab that is currently selected This function is only available in the Standard Curves tab Promega N Display and Manually Adjust Baselines The Display and Manually Adjust Baselines function allows you to set the baseline range for a sample or set of samples Select one or more wells using the well selector then select this button See Section XI D EX Reset Baselines and Amp Thresholds The Reset Baselines and Amp Thresholds function allows you to reset the baseline range and amplification threshold for all samples See Section XLD XI F Amplification Efficiency Calculations The Plexor Analysis Software automatically calculates the equation for the best fit line and determines the R value of the standard curve The R value is a measure of the fit of the data points to a straight line An R value of 1 0 is a perfect fit R values should be close to 1 0 The software also calculates the slope of the standard curve The slope is an indication of the efficiency of the PCR At 10076 efficiency the amount of amplification product doubles with every cycle so C values differ by 1 for each twofold dilution of the template At 100 efficiency the amount of product in
39. e software installation XI C Advanced Options At the File Import screen Step 3 Figure 3 there are two Advanced Options buttons Run Template and Analysis Template These options allow plate configuration and assay parameter information to be saved for reuse during routine experiments Run Template A run template is used to assign sample types sample colors and concentrations of standards Figure 17 If you routinely use the same setup for plates of standard samples and unknowns a run template can be created stored and applied to subsequent runs 1 Select Run Template 2 Assign colors sample types and concentrations to the standards in the Plate Setup tab 3 Use the Sample IDs tab to label your samples Simply select the sample you wish to name and start typing 4 Select Export to save the plate configuration to a rtp file for later use 5 Select OK To import an existing rtp file that contains a saved plate configuration select Import and browse to that file Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 qs Page 53 C P Run Template a EI A Setthe sample types colors target concentrations and sample ID s for each sample using the well selector below rAssignzample types colom and concentrations i e i Propagate Selection Acros
40. ed below Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 ne Page 7 C III B Color Compensation Sample Editing I 1 Sample information can be entered before during or after a run 2 Select Subset Editor Promega 3 Click New to create a new subset Highlight the wells in the plate that contain the color compensation reactions then select Apply 6461TA 4 Select Sample Editor In the Subset pull down menu choose the subset containing your color compensation reactions 5 Under the General tab indicate which are replicate wells Color compensation reactions should be run in replicates of 3 5 Name the samples if desired Subset sev Susser zj Selected Fitter Combinations 523558 5 557 552610 432573 6462TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 8 3r 6 Under the Color Comp tab indicate the dominant color channel for each e color compensation sample using the Dominant Channel pull down menu For No Dye samples Water should be selected as the dominant Promega channel Tr 2 54 Ts eh 7 8 sh io 11 12 AEE EEE 6463TA 7 Select Experiment to return to the experime
41. el select Filter Comb and choose the channel you wish to display The fluorescence data must be exported separately for each color channel Filter Combination SG baei Sad 260 615 670 615 670 558 610 558 610 483 533 4683 533 6484TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA 5 07 Part TM290 Page 29 C 7 To export the amplification data right click on the Amplification Curves chart and select Export from the menu 6485TA 8 Inthe Export chart window select the Data tab Choose Text as the format Include Point Index Point Labels and Header The file should be tab delimited 9 Assign a descriptive name to the file in the Filename box so that it is clear the file contains the amplification data for that specific color channel Choose the box to select a location to save the file then select Export 6486TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 30 VM 10 11 12 15 For multiplex reactions each dye channel must be exported separately by repeating Steps 6 9 In Step 6 use the Filter Comb button to change the color channel being displaye
42. elt temperature and the expected target melt temperature range Failure to set the range for the expected target melt temperature correctly will cause the results to be incorrectly reported in the graph legend and the Reports tab Section IX F For multiplex assays the expected melt temperature range must be adjusted for each dye L Select the PCR Curves tab The default setting for the expected target melt temperature is 90 0 and the default target T range is 1 C Select a well containing a standard reference template or genotyping control sample The Tn for each selected sample will be displayed in a table to the right of the graph Figure 8 The expected target melt temperature and associated target melt temperature range for all samples in this dye channel should be set based on the T of this standard or control sample See Note 1 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Page 38 Printed in USA 5 07 Cuphes Laan Sot for Pedic Diagnostic Lise mplisamen Curves well amp ample D B1 Stndme 125 HO 78B 2601 ves PCR Curves Sample ie 1575 bone Reporte e FAM Tatpat 1 ang Target 2 re RFU ET NI Suandacizt me Tas TYaEDUT var Dd Sundae O44 M1 07587 446 04 Wei D2 ttandaec s54 33 Fes 5 E n1 er F1 Standasd 0 05 20 TRI nde Wie F2 St
43. enzyme activity Confirm the instrument settings and perform a positive control reaction to determine if there is a problem with the Plexor System reagents The primer sequence was incorrect Verify the primer sequence Poor primer design Redesign primers targeting a different region of the gene of interest We strongly recommend using the Plexor Primer Design Software which is available at www promega com plexorresources Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 46 Printed in USA 5 07 X Troubleshooting continued e y Symptoms Causes and Comments Flat amplification curve in the Primer was degraded Use MOPS EDTA Buffer to resuspend Promega amplification curves window and dilute primers Iso dC containing primers are sensitive to no apparent amplification pH Rehydrating or storing the primer in water or a buffer continued with a pH less than 7 0 will result in primer degradation Do not use water to resuspend or dilute primers or make primer mixes Primers may have been synthesized incorrectly Resynthesize primers Primer concentration was incorrect Verify the primer concentration by measuring the absorbance at 260nm The scale of the Y axis was inappropriate If the scale of the Y axis is too broad the change in fluorescence may not be visible Adjust the scale of the Y axis
44. fication curve that did not cross the amplification threshold ren trace MM tanda P standard 5 p a Sample t rmm OO am mem 8 a tinh 8 TE Blandad m Lrdmcss j Samaa mwa aO O a s o on Wurde E T Ye 3 Burm ar Tas a Ye 3 CI MEMESN M ONNNET NER NE Manes 3 83 faded otal TF Faz 2601 vex zu ums Sardana ataj raa i Yer Standard 100 Bam Siardanl 12 5 Standard 12 5 cer ma A Ea ale j i un HHE d a 26 z ABE 1 co temples Standard 26 ID r Star dad 054 Sample 10 Sample 14 Sample t2 5 4E41 T 60602 Ye 2002 E 705 230ED2 Yea vss e elena STII AIAG ite i ea L5 NES geele EEEE L EF eee a i 5055TA Figure 12 The Sample Details tab Thresholds The thresholds report includes the numerical values for the thresholds in the current analysis Figure 13 This information can be used to develop an analysis template for assays where the same or similar thresholds will be used on a routine basis See Section X C for more information about creating an analysis template CT Cumont Amp Thweswd Standard Deviations oo 4100 Mel Cali Threshold aed 20 Content ell Thowmshold Peicentage 250 250 Expected Tm Lowe Threshold 702 738 Expected Tm 792 749 Expected Tm Uppe Threshold 002 75 5056TA Figure 13 The Thresholds tab Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Tol
45. hanges made to the melt threshold line will apply to the entire data set within the same dye channel including those samples that were not selected IX C Adjusting the Y Axes of the Amplification and Thermal Melt Curves Optional The scales of the Y axes for the amplification curve and melt curve in an experiment are determined by the sample that yields the most amplification product i e the sample with the greatest decrease in signal These scales are set for the entire data set The scale of the Y axis can be set manually by double clicking on the Y axis of the graph and entering the new value in the pop up window This change will alter the scale for the entire data set IX D Adjusting the Baseline Region and Amplification Threshold Line Optional The Plexor Analysis Software automatically sets the baseline region for each sample The baseline is set in a flat region of the amplification curve before product accumulation Manual adjustment of baseline is possible See Section XLD for information on display and adjustment of baseline regions The amplification threshold is used to determine the C value for the samples Figure 9 The default amplification threshold is based on the variation noise in the baseline regions of all samples It is determined by taking the mean and standard deviation of all RFU values in the baseline regions and setting the threshold to 10 standard deviations below the mean Optional If desired
46. icieney CATCUIBOTS coerceri inset receta th recun FE ERR oe ERR 99 eE RR 99 I Description The Plexor qPCR and qRT PCR Systems are compatible with a variety of real time PCR instruments Data from these instruments can be analyzed with one dedicated software program the Plexor Analysis Software This manual includes instructions and thermal cycling conditions specific for use of the PlexorTM qPCR System Plexor One Step qRT PCR System and Plexor Two Step qRT PCR System with the Roche LightCycler 480 System Instructions are included for instrument setup data transfer from the instrument to the Plexor Analysis Software and data analysis II Plate Preparation and Amplification Detailed instructions describing assay setup are provided in the Plexor qPCR System Technical Manual TM262 Plexor One Step qRT PCR System Technical Manual TM263 or Plexor Two Step qRT PCR System Technical Manual TM264 When using the Plexor qPCR System for the first time we recommend programming the thermal cycling conditions and checking that the instrument is compatible with the dyes used and is configured for those dyes before assembling the reactions so the reactions are not kept on ice for prolonged periods of time Once you are familiar with the programming process the instrument can be programmed after reaction assembly Materials to Be Supplied By the User LightCycler 480 multiwell reaction plate e centrifuge compa
47. l Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 44 Printed in USA 5 07 Baseline Regions The baseline regions report includes the numerical values for the C cycle number used in each sample Figure 14 e Run Info The run info report includes the information from the data import Promega Figure 15 Import Files The Import Files report includes information on the data import files Figure 16 CER as 710 TETE 230 ee Ms x ja unen3 Bs x40 18 35 5057TA 5242TA 5508TA Figure 16 The Import Files tab Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 d Page 45 IX G Saving and Printing the Analysis File 1 The Plexor Analysis Software saves the analysis as an aan file The current analysis can be saved at any time by selecting Save Analysis File aan in the File menu 2 Selected wells can be exported into a new analysis file In the File menu select Export Selected Wells as New Analysis File aan 3 The analysis screen can be printed or saved as a screenshot In the File menu select Save a Screenshot png or Print a Screenshot 4 ARunTemplate and Analysis Template from an existing analysis can be exported and used in future analyses Section XI C X Trouble
48. leach solution before and after use Use new reagents and solutions Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 51 Promega X Troubleshooting continued Symptoms Causes and Comments No amplification in the positive The RNA template used in the Plexor qRT PCR Systems was control reaction continued degraded RNA storage conditions are very important Store RNA template at 70 C in single use aliquots to minimize the number of freeze thaw cycles Once thawed keep RNA on ice Always use nuclease free commercially autoclaved reaction tubes sterile aerosol resistant tips and gloves to minimize RNase contamination Reactions were assembled incorrectly Repeat the experiment and assemble reactions as described in the Plexor qPCR System Technical Manual TM262 the Plexor One Step qRT PCR System Technical Manual TM263 or the Plexor Two Step qRT PCR System Technical Manual TM264 Unable to import data An error The data has been altered after export from the real time PCR like Expecting NEWLINE instrument software Any alteration of this data is likely to found or Unexpected Token change the formatting and can cause import errors Do not Error is encountered open the exported files with other software programs Data display in the Plexor Analysis Be sure that
49. n results from a dilution series of the standard reference template are used to generate a standard curve This standard curve can be used to determine the concentration of unknown samples A standard reference template with any unit of concentration or amount can be used to generate the standard curve In general copy number or mass is used but other units that are appropriate for your experiment such as plaque forming units or dilution factors from a known stock can be used Samples for generating the standard curve must be designated as standards Section IX A For multiplex assays standard curves must be generated for each dye label 1 Select the desired standard samples and the samples you want to quantify Select Add Standard Curve to generate a standard curve ps Add Standard Curve Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 5 07 Page 41 4 Select the Standard Curves tab to view the standard curve Figure 10 The default display shows the log concentration on the Y axis and the cycle threshold on the X axis Alternatively the standard curve can be displayed with the cycle threshold on the Y axis and the log of the concentration on the X axis Figure 11 To do so select the Standard Curves tab to view the standard curve In the Edit menu select Flip Std Curve Axes See
50. ndaed MO 278 JA 10600 Yes y Q 30x 10 44 3 Sunded Y 270 784 10802 ves R 0983 id Sample 203 T03 20EQ ras B Samples 270 N2 95001 vu Sampla i 74 TRI 13603 ar Sindsd 125 30 789 1260 Ter funded 125 SOR 788 12B0 Yes B3 Bude i275 3U5 Tee 12bU ves Ji Sampled 20 7802 1004 ver Sampie 5 3180 TUZ B TEOD ar sample 32 0 7802 t0EO4 Yar Bandas 2 5 mn Tes 5E0 Yas Bundud2s 320 702 25E00 ves Standard 2 4 X28 Tt SSR vu Sampla T M13 TUT 10600 rar Sample Mo 702 BAED ve tamps Ma Th SFE rwr Btandaed DIA 1 TMs amp 4E Tar Bland aedi 053 Ma ToD BAEO1 v Buai 354 792 SADI ver Sample 10 304 74A 5BEUT Tar ample 13 7 T05 GEM Tw Bampa 12 302 705 20E Yas Biacwland C 18 rs T7282 TREO1 Yei Btandaed C me 5 i gkOT Tar Stndaed Cie 253 THs BED Ta lampia 13 Se 32IEM Tu Sample 14 288 728 22602 Yes Sample 15 MA TOS 25B Yn Bhared aedi Dc at Tul Z E Yei SundmdOD2 304 702 20802 ver Stand aed Cue 3 Ta 206 02 Yar I p 176p 7 3 79 7 x sp pard 2a 20 a Fi du 34 Lo ocho ho ho A hoch ho h oh ho h cho h oh h oh Roch koh ho hoch ho ho ch Oh Rok d 1 Binig LU 3855 NS Seo vasf T BSndaed DOO u Tu5 DEUT ws oX bar NTC does not have a valid CI vale 3 Punios Wo 793 6 0802 Ter lt P H2 NTC does not have a valid Cl value RERO C DECOAEE Tn 7 39H NTC does not have a valid CI wakia S Range 300 702 aim ves IB Bampa ii mo 70I 2260 Tes 61 aa x 505
51. nspecific amplification products Design new primers to minimize the synthesis of nonspecific amplification products Reactions were contaminated with target DNA or RNA Clean pipettes and workstations with a mild bleach solution before and after use Use new reagents and solutions Use positive displacement pipettes or aerosol resistant tips to reduce cross contamination during pipetting Use a separate work area and pipettes for pre and postamplification Wear gloves and change them often No amplification in the positive No amplification or poor amplification See causes and control reaction comments for Flat amplification curve in the amplification curves window no apparent amplification above Verify that the thermal cycling program and data collection settings are correct Section IV V or VI Instrument setup problems can cause amplifications to fail Consult the instrument manufacturer s user s manual for more information about potential instrument problems The Plexor Master Mix may have lost activity Be sure to store the Plexor qPCR and qRT PCR Systems at 20 C to avoid loss of enzyme activity Confirm the instrument settings and perform a positive control reaction to determine if there is a problem with the Plexor System reagents The RNA template used in the PlexorTM qRT PCR System was contaminated with ribonuclease RNase Take precautions to prevent RNase contamination Clean workstations and pipettes with a mild b
52. nt window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 d Page 9 CQ II C Color Compensation Reaction Setup v You will need a separate color compensation reaction dedicated to each individual Promega dye in your multiplex as well as a reaction with no dye Table 2 Each of these color compensation reactions should be run in replicates of 3 5 The same Plexor primers that will be used in your experimental samples can be used to set up the color compensation reactions The primers in the color compensation reactions should be used at similar concentrations to those in the experimental reactions When using Plexor primers it is not necessary to add template to the color compensation reactions Note The volumes listed in Table 2 are for use with a 96 well reaction plate When using a 384 well reaction plate reaction components should be scaled down to a final volume that is compatible with that plate Plexor reaction components have been used successfully in reaction volumes as low as 5ul In a 384 well reaction plate 10ul final volumes are commonly used Table 2 Color Compensation Reaction Setup Reaction Components Plexor Dye Reaction No Dye Reaction Blank 2X Plexor Master Mix PASA 12 5ul Plexor primer 1u MOPS EDTA Buffer 11 51 12 530 Total Volume 25ul 25ul 1 Setu
53. on JO 2 minutes 1 cycle Denaturation 5 seconds Annealing and 40 cycles Extension 35 seconds Denaturation IDC 5 seconds 1 cycle Melt Temperature 50 C to 95 C 2 5 acquisitions per C Curve Instrument Cool 50 C 30 seconds 1 cycle Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 qs Page 13 C 1 Open the LightCycler 480 software 2 2 Select New Experiment in the startup screen peg LCAN 1243 Stamidlry ni ENAN Database My Computer Traceable Mind Overview Urar Symi Adnin mg i oe ta o Sra 6453TB 3 Select the appropriate detection format from the pull down menu The detection format specifies which fluorescent channels will be read created in Section II A Wi Hono Color Hydrolysis Proce 1 Multi Color Hydrolysis Probe M Bono Color xybPrcbe Multi Color HWHybFrobe by FAH COSB0 CRO35 QETO 6467TA 4 Enter the reaction volume Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 14 5 07 5 The cycling will consist of four programs Program the instrument by C adding the program names in the top window Select the icon to add a program In
54. oration 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 50 Printed in USA 5 07 X Troubleshooting continued CO Symptoms Causes and Comments Promega Slope less than 0 2 No amplification or poor amplification See causes and inefficient amplification comments for Flat amplification curve in the amplification curves window no apparent amplification above Nonspecific amplification can become a problem in later amplification cycles with samples containing small amounts of target template Decrease the number of amplification cycles Poor primer design Design new primers Annealing temperature was too high Design new primers with melting temperatures of 60 C We strongly recommend using the Plexor Primer Design Software Annealing temperature was too high Optimize the annealing temperature Amplification in no reverse Contaminating DNA sequences related to the RNA template transcription control for the were present in the RNA preparation Treat the RNA Plexor qRT PCR Systems template with DNase to remove contaminating DNA Design new primers to span introns to avoid amplification of contaminating genomic DNA Nonspecific amplification occurring in reactions that contain a low number of copies of the template Assemble reactions on Ice Decrease the number of amplification cycles to reduce accumulation of no
55. ose the appropriate detection format from the pull down menu d3Y8R Green I Simplefroba Arora jHono Color Hydrolysis Probe NT Z lmulti Color Hydrolysis Probe Hono Color HybProbe Hulti Color HyhProbe FAN COSBO CRE35 Q6 TO 6456TA 9 Enter the reaction volume Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 oe Page 5 C 10 The cycling program should mimic a typical PCR This cycling will consist So of four programs Program the instrument by adding the program names Promega in the top window Select the icon to add a program In the Program Name section name the first program of this experiment Denature or a similar title In the Analysis Mode pull down menu select None 11 With the Denature program name selected change Program Temperature Targets to the values indicated below Add new program Add new step to selected program 6457TA 12 Next to Program Name select the button to add a program Name the second program of this experiment PCR or a similar title Enter the number of amplification cycles In the Analysis Mode pull down menu choose Quantification 13 With the PCR program name selected change Program Temperature Targets to the values indicated below To add new steps select the
56. p the color compensation reactions on ice in a LightCycler 480 multiwell reaction plate 2 Mix each color compensation reaction thoroughly 3 Cover the reaction plate with an optical adhesive cover using the applicator Centrifuge briefly to collect contents at the bottom of each well 4 Load the reaction plate into the LightCycler 480 and immediately press Start Run to begin thermal cycling Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 10 SUP IILD Creating a Color Compensation Analysis File C 2 1 When the run is complete select Analysis 2 Inthe Analyses pull down menu choose Overview 3 Inthe Create new analysis list select Color Compensation Create new analysis l bs Quant znd Derivative Max jabs Quant Fit Points o ion 6464TA 4 When the Create new analysis pop up window appears select the sample subset containing your color compensation reactions from the Subset pull down menu Select OK Create new analysis F Color Compensation EJEJETEIBIESESES i OK button 6465TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 aa Page 11 C 5 Select Calculate to perform
57. r for the dye set used Decrease the primer concentration e g 0 1uM Primer pairs in a multiplex reaction can interact to form undesired amplification products Perform a BLAST search to reveal regions of identity with undesirable target sequences Label the primer with the lowest homology to other sequences Alternatively design new primers using the Plexor Primer Design Software which is available at www promega com plexorresources Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 y0 Page 47 CQ X Troubleshooting continued v Symptoms Causes and Comments Promega Broad melt curve orashoulderon Pseudogenes and polymorphic genes may exist Perform a the melt curve BLAST search of the target sequence When designing primers choose target sequences that have the fewest regions of identity with pseudogenes and polymorphic genes Check for signal bleedthrough Calibrate the instrument as instructed in Section III or per manufacturer instructions Decrease the primer concentration e g 0 1uM Be sure the thermal cycler was programmed correctly Section IV V or VI No melt curve observed in the Poor amplification See causes and comments for Flat melt curve window amplification curve in the amplification curves window no apparent amplification above Problems with data export or in
58. s please contact Promega Corporation Licensing 2800 Woods Hollow Road Madison WI 53711 or EraGen Biosciences Corporate Licensing 918 Deming Way Suite 201 Madison WI 53717 Phone 608 662 9000 Fax 608 662 9003 This product is designed and sold for use in the multiplex PCR process covered by U S Pat No 5 582 989 and Canadian Pat No 1 339 731 A limited license has been granted under the patent to use only this amount of the product to practice the multiplex PCR process and is conveyed to the purchaser by the purchase of this product 2007 Promega Corporation All Rights Reserved RNasin is a registered trademark of Promega Corporation ImProm II and Plexor are trademarks of Promega Corporation LightCycler is a registered trademark of Roche Diagnostics GmbH Macintosh is a registered trademark of Apple Computer Inc Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277
59. s Dyer FAM Target JOE Target2 258 LHounmouqenu ss fo o o o o oO lo o fo ga Oo jo ooo lo 5060TA Figure 17 A Run Template Analysis Template and Definition of Analysis Functions The analysis template is used to optimize the analysis settings for the experiment If you routinely perform reactions with the same analysis conditions an analysis template can be created stored and applied to subsequent runs These settings can be exported as a ntp file then imported for subsequent experiments A description of the functions for each setting follows 1 Select Analysis Template 2 Enter the desired values for the analysis defaults for each dye used Figure 18 Note Descriptions of the analysis details are provided below 3 Select Export to save the default settings to a ntp file for later use 4 Select OK To import an existing ntp file that contains the saved default settings select Import and browse to that file Default Amplification Threshold RFU Baseline Noise Standard Deviations The Plexor Analysis Software has a user definable amplification threshold that determines the RFU value at which sample cycle thresholds are called This value is based on the variation noise in the baseline regions of all samples and is determined by taking the mean and standard deviation of all RFUs in baseline regions The threshold is set a specified number of standard deviations below the mean
60. s the Instrument Select either 96 Well Block or 384 Well Block as is appropriate for your sample run Step 1 Assay Setup In order ja define Ine assay vou wish to import please specify the following e parameters Required dade Assay Name Please enter the name of this assay Step 2 instrument Please select the supported instrument for this assay Roche LightCyder 480 96 Well Block Roche LighhCycler 480 334 Well Block BioRad iCyder iQ TBiofad Cyder iQ via paste Cancel 6490TA Figure 1 The Assay Setup screen 5 Select Add Target for each fluorescent dye used in your assay For each dye assign a target name enter the dye name and indicate that there is amplification data and dissociation melt data to be analyzed for that dye The name of the dye must be the same as that in the original exported data file Note For frequently run assays a template with the target information and dyes can be saved Section XI C 6 Select Next Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 y0 Page 33 7 Enter information specific to your experiment in the Run Info screen Figure 2 Details date notes title name of the person performing the experiment etc can also be entered in the provided windows Run Info
61. same dyes and similar cycling conditions can be analyzed using this color compensation file Notes The Roche LightCycler 480 instrument should be programmed before preparing the reagents e The color compensation file can be generated after the experimental run and applied to the multiplex experiment before final analysis e A list of LightCycler compatible dyes is available at www promega com plexorresources II A Thermal Cycling Program for Color Compensation The thermal cycling program is shown in Table 1 Primers designed using the Plexor Primer Design Software have an annealing temperature of approximately 60 C Table 1 Color Compensation Thermal Cycling Program Step Temperature Time Number of Cycles Initial Denaturation IL 2 minutes 1 cycle Denaturation 5 seconds 40 cycles Annealing and Extension 35 seconds Denaturation 95 C 5 seconds 1 cycle Melt Y Temperature 50 C to 95 C 2 5 acquisitions per C Curve Instrument Cool 50 C 30 seconds 1 cycle Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 5 07 Page3 6453TA 1 Open the LightCycler 480 software 2 Tocreate a new dye set detection format select Tools in the startup screen The detection format specifies the channels in which fluorescence will be read ee a A Seales Database
62. samples within the PCR plate Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Page 36 Printed in USA 5 07 Unknown No template control Standard sample The concentration is entered in a pop up window following designation of the well as a standard e m Selecting the wells and choosing the Create Dilution Series icon can automatically create a titration curve across several wells Positive control Color assignment Lm W Figure 5 The icons used to define samples in the Plexor Analysis Software PCR Cumt Sample Ies tiani mapaes 5 aiat i35 i 1 LET r Fr l m n is aT i 5048TA Figure 6 The Sample IDs tab Notes 1 Sample definitions color selection and concentration of standards will be entered for all dyes To define samples separately in each dye uncheck Propagate Selection Across Dyes in the Edit menu 2 A sample or set of samples can be permanently deleted from a Plexor analysis Go to the PCR Curves tab and select the samples Use the delete key or select Remove Selected Wells in the Edit menu 3 All samples defined as standard reference templates must be assigned a concentration Concentrations may be entered in standard format 0 01 0 1 1 10 100 1000 etc or scientific format 1e 2 1e 1 1e0
63. shooting Symptoms Flat amplification curve in the amplification curves window no apparent amplification Causes and Comments Be sure that the reactions were assembled correctly See the Technical Manual supplied with the Plexor Systems Template was degraded or of insufficient quantity Verify the integrity of the DNA or RNA template by electrophoresis Repeat the DNA or RNA purification if necessary Add RNasin Ribonuclease Inhibitor to the reaction to inhibit a broad spectrum of RNases Amplification inhibitor was present in the DNA or RNA template Reduce the volume of template in the reaction Repeat the DNA or RNA purification if necessary Add the template in question to the positive control reaction A significant increase in the C value or no amplification in the positive control reaction indicates the presence of inhibitors in the template Thermal cycler was programmed incorrectly Verify cycle times and temperatures Section IV V or VI Data collection settings were incorrect Data collection must occur during the extension step The extension time must be sufficient for data collection Verify the data collection settings The wrong dye or detector was selected or the dye was incompatible with the instrument Be sure the selected detectors are appropriate for the fluorescent dyes used The Plexor Master Mix may have lost activity Be sure to store the Plexor qPCR and qRT PCR Systems at 20 C to avoid loss of
64. sly by highlighting multiple capillaries and using the Create Dilution Series function NS Add Standard Curve shortcut d The Add Standard Curve function fits the experimentally measured C values and user entered concentration values for standard samples to a straight line using the least mean squares method It will calculate the concentrations of unknown samples positive control reactions and no template control reactions from their measured C values using the equation for the best fit line Any sample with a concentration of N A on the report or elsewhere did not cross the cycle threshold so the concentration of that sample cannot be calculated Select all of the samples you wish to use as standard samples as well as all other samples for which you wish to calculate concentrations Choose Add Standard Curve from the Edit menu Type d or select the Add Standard Curve icon on the toolbar You may create as many standard curves as you wish for a single set of data but no sample can be used to generate more than one standard curve It is not possible to Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Page 58 Printed in USA 5 07 add samples to an existing standard curve but a new curve can easily be C constructed with a new selection This action will remove the existing standard e curv
65. software select Sample Editor Eres ad ed pi ae B SSI EAE E i r 4 oo Iz HHHH 6472TA 3 Under the General tab indicate which are replicate wells Name the samples if desired 4 Click Experiment to return to the Experiment window 5 Load the reaction plate into the LightCycler 480 and immediately press Start Run to begin thermal cycling Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM290 oe Page 27 Promega VII Data Export from the Roche LightCycler Data Analysis Software Before the data can be analyzed using the Plexor Analysis Software the data must be exported from the LightCycler 480 software Two txt files must be exported for each color channel used one with the amplification data and one with the melt dissociation data Be sure to use a descriptive name when naming these files so that it is clear which file contains the amplification data and which file contains the dissociation data while still indicating that the files are related Preliminary analysis must be performed using the LightCycler software 1 Open the experiment to be analyzed Pnalyses overvies O 2 Click on Analysis on the toolbar reate new an mE Abs Quant and Derivat ive fax 3 To analyze the amplification data select Calar Compensation Overview in the
66. strument analysis have occurred Review the instructions for data export and instrument setup Data collection settings were incorrect Verify the thermal cycling program and data collection settings are correct Section IV V or VI Incorrect files were imported Be sure to import the proper files containing related amplification data and dissociation data Instrument was programmed incorrectly Verify the thermal cycling program is correct Section IV V or VI Variability in signal among Calibrate your pipettes to minimize variability in pipetting replicate samples Small volumes are difficult to pipet accurately Do not pipet volumes lt 1pl dilute the template so larger volumes are pipetted Some variation is normal A difference of 1 2 cycles for the C values is within the normal variation associated with an exponential amplification reaction There will be statistical variation in the amount of template in a reaction with targets present at low copy number Poisson distribution predicts difficulty associated with reliable detection of very dilute samples with few target molecules Mixing was inadequate Vortex reagents to mix well prior to pipetting Use reaction plates recommended by the instrument manufacturer Instrument was improperly calibrated Calibrate instrument as instructed by the manufacturer Thermal cycling conditions were suboptimal Optimize the annealing temperature Thermal cycling conditions were suboptimal
67. strument by adding the program names in the top window Select the icon to add a program In the Program Name section name the first program of this experiment Reverse Transcription and denaturation inactivation or a similar title In the Analysis Mode pull down menu select None With the Reverse Transcription and denaturation inactivation program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window i 7 Ari 6473TA Next to Program Name select the button to add a program Name the second program of this experiment PCR or a similar title Enter the number of amplification cycles In the Analysis Mode pull down menu select Quantification With the PCR program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window With the 60 C target temperature selected change the acquisition mode to Single Nelting Curves Quanrcification T 6474TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 20 Printed in USA 5 07 9 Next to Program Name select the button to add a program Name C the third program of this experiment Melt or a similar
68. the amplification threshold may be reset to change the sensitivity in detecting the amplification product Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Page 40 Printed in USA 5 07 1 To adjust the amplification threshold based on a selected set of samples C highlight the desired samples in the well selector In the Edit menu 9 select Set Amp Threshold From Selected Samples Enter the number of standard deviations of the background within the baseline region to use The default is 10 standard deviations Promega 2 To manually adjust the threshold place the cursor over the threshold and drag the line to the desired location Alternatively double click on the threshold and enter the desired value Note Changes made to the amplification threshold will apply to the entire data set within the same dye channel including those samples that were not selected The baseline and threshold reset button will reset the amplification R threshold to the default using all samples Duplex 1 aan hot for Medical Diagnostic Use PCR Curr Sample Ibs pisna are CHmeec Reports FAM Target ine Targeta A umplilizanon Curvas Amplification Threshold AFL 5052TA Cycle Figure 9 The amplification and melt thresholds IX E Generating a Standard Curve Optional Amplificatio
69. the color compensation analysis For each A filter combination the Raw Data chart shows the raw fluorescence data while the Compensated Data chart shows the fluorescence after the Promega color compensation has been applied kolor Compensation fcr New Subset 1 zi 6466TA 6 Select Save CC Object to save this color compensation analysis to the database This color compensation file can now be applied to data in another experiment Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Printed in USA Page 12 UM IV Instrument Setup and Thermal Cycling for qPCR and Two Step qRT PCR e These instructions describe instrument setup and thermal cycling conditions for DNA or cDNA quantitation using the Plexor qPCR or Plexor Two Step qRT PCR Promega System Thermal cycling programs described in this manual are optimized to work with primers designed using the Plexor Primer Design Software The Plexor Primer Design Software can be accessed at www promega com plexorresources IV A Thermal Cycling Program The thermal cycling program is shown in Table 3 Primers designed using the Plexor Primer Design Software have an annealing temperature of approximately 60 C Table 3 qPCR and Two Step qRT PCR Thermal Cycling Program Step Temperature Time Number of Cycles Initial Denaturati
70. the display settings for the computer are set to Software appears abnormal 32 bit color rather than 16 bit color when using the Plexor the screen appears compressed Analysis Software lines are replaced with dots etc Genotyping Miscalled known Poor primer design Redesign primers We strongly recommend heterozygous samples Product using the Plexor Primer Design Software which is available formed with only one of the two at www promega com plexorresources genotyping primers The annealing temperature was too high or too low Optimize the annealing temperature Genotyping Miscalled known Poor primer design Redesign your primers We strongly homozygous samples Product recommend using the Plexor Primer Design Software which formed signal decrease with is available at www promega com plexorresources both primers The annealing temperature was too high or too low Optimize the annealing temperature Genotyping Miscalled known The primer sequence was incorrect Verify that the primer homozygous samples Product sequence is correct formed only with the mismatched primer but not with the matching Genotyping primer 1 and primer 2 were switched Verify primer that the correct primer was used Genotyping No call Add more template Redesign primers See comments for Flat Amplification Curve Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 27
71. tible with a 96 well plate e optical adhesive covers and applicator 1 After the amplification reactions have been assembled cover the reaction plate with an optical adhesive cover using the applicator Centrifuge briefly to collect contents at the bottom of each well Note Keep the plate on ice during reaction setup and programming of the thermal cycling conditions 2 Program the Roche LightCycler 480 System The proper thermal cycling conditions and instructions for programming the instrument are provided in Section III generating color compensation files Section IV qPCR and two step qRT PCR assays Section V one step qRI PCR assays and Section VI genotyping assays Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM290 Printed in USA Page 2 5 07 III Generating Color Compensation Files e y For multiplex assays a color compensation file cc object must be created and p applied to multicolor data to allow proper interpretation To generate the color mega compensation file labeled Plexor primers are used as dye calibrators in an initial color compensation cycling experiment The color compensation cycling experiment contains separate color compensation reactions corresponding to each individual dye in your multiplex as well as a blank reaction with no dye Any subsequent runs performed with the
72. title In the Analysis Mode pull down menu select Melting Curves Promega 10 With the Melt program name selected change Program Temperature Targets to the values indicated below To add new steps select the button in the lower window With the 95 C target temperature selected change the acquisition mode to Continuous and change Acquisitions per C to 2 5 6475TA li dene 11 Next to Program Name select the button to add a program Name the fourth program of this experiment cooling or a similar title In the Analysis Mode pull down menu select None 12 With the cooling program name selected change Program Temperature Targets to the values indicated below 6476TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Partt TM290 oe Page 21 C V B Sample Editing 2 1 Sample information can be entered in the LightCycler 480 software Promega before during or after your experiment is run Alternatively sample names can be entered during the data analysis step using the Plexor Analysis Software 2 To enter sample information using the LightCycler 480 software select Sample Editor 6472TA 3 Under the General tab indicate which are replicate wells Name the samples if desired 4 Click Experiment
73. tton indicates the color channel being displayed To view each color channel select Filter Comb and choose the channel you wish to display The fluorescence data must be exported separately for each color channel To export the melt data right click on the Melting Curves chart and select Export from the menu Melting Curves 6489TA In the Export chart window select the Data tab Select Text as the format Include Point Index Point Labels and Header The file should be tab delimited Assign a descriptive name to the file in the Filename box so that it is clear the file contains the melt data for that specific color channel Choose the box to select a location to save the file then select Export For multiplex reactions each dye channel must be exported separately by repeating Steps 15 18 In Step 15 use the Filter Comb button to change the color channel being displayed in order to export data from each of the color channels If analysis will be done on a separate computer save the files on removable media or an accessible network location These exported files are now ready for use with the Plexor Analysis Software Note The Plexor Analysis Software will use the greatest change in signal throughout the imported data set to determine a signal threshold This may affect sensitivity of assays with lower signal if multiple types of assays in the same color are being run simultaneously
74. ycles before the decrease in fluorescence and in an area where the baseline is flat The C values for selected samples are displayed in the table to the right of the graph The C value may change when the limits are changed See Notes 1 and 2 If necessary adjust the lower limit to a region that creates the flattest baseline given the selected upper limit Optional The amplification threshold is based on noise within the baseline region for all of the samples When manual baseline adjustments are complete consider recalculating the amplification threshold for all samples Select all samples and in the Edit menu select Set Amp Threshold from Selected Samples Section IX D See Notes 3 and 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM290 Page 56 Printed in USA 5 07 Notes C 1 e A maximum upper limit of 35 cycles can be used for samples without a C value e g no template control Promega 2 Samples with similar C values can be adjusted simultaneously by highlighting multiple wells 3 The baselines for all samples can be reset to the automatic setting by selecting the Reset Baselines and Amp Thresholds icon cx 4 Toreset the baselines for a selected set of samples select the samples in the well selector and in the Edit menu select Set Baselines for Selected Samples

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