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Plexor(TM) Systems: Instrument Setup and Data Analysis for the Bio

1. Run Time micromoles SA Central I Define a dilution series IV Scientific Notation Define sample Ai and replicates Standard Quantity Sample Identifier Data Analysis 5226TA Figure 3 The Whole Plate Loading tab in the Edit Plate Setup window Samples Whole Plate loading Select and load fluorophores 1 Select or deselect a fluorophore 2 Assign a color 3 Enter plate notes ROX 575 SYBR 490 TAMRA 530 TAMRA 548 TET 490 _ TET 530 v Available Filter Wheels FilterSeta Define sample wells 4 Load well fluorophores by clicking to select a Fluor pen and then clicking wells that contain a defined sample 5227TA Figure 4 The Select and load fluorophores tab in the Edit Plate Setup 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 Part TM270 Printed in USA Page 6 9 05 C 12 The Run Prep window will open Figure 5 Enter a sample volume of A 25ul and check that the Experimental Plate radio button is selected under the Select well factor source option unless you are using an Promega external well factor plate as described in Section IX G 13 Place the PCR plate into the instrument and immediately begin thermal cycling by selecting Begin
2. 5239TA Figure 25 The Assay Setup screen 27 Select Bio Rad iCycler iQ as the Instrument 28 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 Note For frequently run assays a template with the target information and dyes can be saved Section IX C 29 Select Next 30 Enter information specific to your experiment in the Run Info screen Step 2 Figure 26 Details date notes title name of the person performing the experiment etc can be entered in the provided windows 31 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 9 05 Part TM270 Page 25 Please fill in the details below regarding your run Promega pate Experiment Title Operator Name Date sD _ i Septembar 14 2008 aaa peas Cancel lt Back Next gt 5238TA Figure 26 The Run Info screen 32 Use the File Import screen Step 3 Figure 27 to specify the Excel data files exported from the instrument see Steps 1 23 Use Browse to locate the appropriate exported amplification and melt dissociation data files When analyzing data with the Plexor Analysis S
3. table View Quantities Identifiers View Post Run Data Data Analysis Operations Analyze Data Use External Calibration file PCR Amplitude Cycle Standard Curve Melt Curves EE N Melt Peaks 5235TA Figure 22 The View Post Run Data in the Library module 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 TM270 Page 22 Printed in USA 9 05 10 Copy the data from the iCycler iQ software C amp 11 Open a new Excel spreadsheet select well A1 and paste the values into the spreadsheet Promega 12 Save this spreadsheet as a txt tab delimited file with a name that describes the dye and indicates that it is an amplification data set i e Plexor FAM amp txt 13 Select a different fluorophore pen and repeat steps 7 12 for each dye used 14 To export the melt data go to the Melt Curve tab of the Data Analysis module 15 Highlight a fluorophore pen from the Select a Fluorophore list 16 Once the melt curve graph is shown select the dF dT vs Temperature tab to display the melt derivative 17 Right click on the melt curves graph and select Display Data This will display a table in a small frame called Data Display above the melt curves graph 18 Left click on the cell labeled Well Temp to select all data in
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 Printed in USA Part TM270 2 0 Page 31 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 VIII for more information about possible causes Changes 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 VILC 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 eraph and entering the new value in the pop
5. ard 0 54 ard 0 54 ard 0 54 e 10 ed ed2 ard 0 18 ard 0 18 ard 0 18 13 e 14 e 15 ard 0 06 ard 0 06 ard 0 06 e 16 e17 e 18 26 27 28 29 230 31 32 33 34 35 Ct Cycle Threshold Figure 34 The Standard Curves tab A standard curve with the log concentration on the Y axis and the cycle threshold on the X axis PCR Cuwes Sample Ibs Standard Curves Reports FAN Target Curve 1 Standard Curve 1 y 3 2 fx 34 34 Standard 0 02 Standard 0 02 Standard 0 02 Sample ID Standard 100 Standard 100 Standard 100 z R 0 993 Ct Cycle Threshold H3 NTC does not have a valid Ct value R lt H2 NTC does not have a valid Ct value lt H1 NTC does not have a valid Ct value Log Concentration Figure 35 A standard curve with the cycle threshold on the Y axis and the log concentration on the X axis Note Samples that do not cross the amplification threshold such as the no template Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp ed e2 a3 ard 12 5 and 12 5 ard 12 5 e4 a5 eG and 2 5 and 2 5 ad 2 5 aT e8 eg ard 0 54 ard 0 54 ard 0 54 e 10 edi e12 ard 0 418 ard 0 18 ard 0 18 13 e 14 e 15 ard 0 06 ard 0 06 ard 0 06 e 16 a 17 e 18 Standard 0 02 Standard 0 02 Standard 0 02 controls
6. 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 Plexor qRT PCR System was contaminated with ribonuclease RNase Take precautions to prevent RNase contamination Clean workstations and pipettes with a mild bleach 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 Part TM270 9 05 Page 43 VIII 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 Plexor One Step qRT PCR System Technical Manual TM263 or Plexor Two Step qRT PCR System Technic
7. Carefully seal the plates to avoid evaporation Aberrant fluorescence can be caused by writing on plates contamination fingerprints etc Do not write on the plate Use caution when handling plates Wear gloves Do not place plates on surfaces that might be contaminated with a fluorescent material If you suspect contamination of the benchtop thermal cycler block or any other area clean it thoroughly 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 TM270 Page 42 Printed in USA 9 05 VIII Troubleshooting continued CQ A Symptoms Causes and Comments Slope less than 0 2 No amplification or poor amplification See causes and Promega 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 is too high Design new primers with melting temperatures of 60 C We strongly recommend using the Plexor Primer Design Software Annealing temperature is too high Optimize the annealing temperature Amplification in no reverse Contaminating DNA related to the RNA template is present transcrip
8. Printed in USA Part TM270 9 05 Page 29 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 31 You must enter the initial concentration of the series the dilution factor and whether the series is increasing or decreasing 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 Mertical Series Horizontal Series Starting Concentration fi Dilution Factor fio ir Increasing C Decreasing Apply Cancel 5049TA Figure 31 The Assign Dilution Series screen VILB Adjusting the Expected Target Melt Temperature Set the expected target melt 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 VIL F For multiplex assays the expected melt temperature range must be adjusted for each dye 1 Select
9. 1 aan Not for Medical Diagnostic Use Tab Selection PCR Cumes Sample bs iar dsrc Cure Repos FAM Target 1 soe Target2 Tools B1 13 B2 D1 D2 Amplification J Jrves Window Melt Curves Window ARFUYyET Well Selector 5046TA Figure 28 The PCR Curves tab of the analysis desktop The amplification curves window melt curves window and well selector are indicated VILA Sample Definition 1 Use the well selector which is shown in Figure 28 to select and define each well or group of wells Choose one of the icons shown in Figure 29 to define the samples See Notes 1 5 2 To assign sample names select the Sample IDs tab Figure 30 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 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 Part TM270 Page 28 Printed in USA 9 05 Gm gis C 1062 A e Unknown No template control Standard
10. Protocol Edit Plate Setup View Quantities Identifiers Run Prep Begin Run Sample volume 25 ul Select run purpose Select well factor source PCR Quantification a T Experimental Plate C Pure Dye Calibration C Well Factor Plate Running plate setup contains no wells with the Pure Dye sample type Run Time Central 5228TA Figure 15 The Run Prep 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 Part TM270 Printed in USA Page 16 710 VI Data Export from the iCycler iQ and Import into the Plexor Analysis Software CQ A Data may be transferred from the iCycler iQ to the Plexor Analysis Software in P two ways The recommended method is to directly copy and paste data from the mega iCycler iQ software into the Plexor Analysis Software Section VI A However this method requires that the Plexor Software be installed on the instrument workstation Alternatively data may be copied and pasted the iCycler iQ into Microsoft Excel The Excel files are saved and then imported into the Plexor Analysis software Section VIB VI A Pasting Data Directly into the Plexor Software This method requires that the iCycler iQ Software and the Plexor Analysis Software both be open on the instrument workstation The Plexor Analysis Software Cat A40
11. 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 primer sequence is 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 TM270 Page 38 Printed in USA 9 05 VIII Troubleshooting continued CQ A 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 i
12. 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 primer Genotyping primer 1 and primer 2 were switched Verify that the correct primer was used Genotyping No call Add more template Redesign primers See comments for Flat amplification curve in the amplification curves window no apparent amplification 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 TM270 Page 44 Printed in USA 9 05 IX Appendix C IX 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 IXB 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 in
13. are listed in the graph as not having a valid C value Ct 28 0 27 8 27 6 26 3 27 8 27 4 31 0 30 8 30 5 31 0 31 6 341 0 33 0 32 9 32 9 34 3 35 0 34 4 35 1 35 3 35 4 38 4 37 7 30 3 37 5 36 65 36 3 26 4 26 6 26 5 33 5 39 0 33 6 26 5 26 0 26 6 40 1 30 4 39 2 26 0 278 275 25 3 27 8 27 4 31 0 30 8 30 5 34 0 31 6 34 0 33 0 32 9 32 9 34 3 35 0 34 4 35 1 35 3 35 4 38 4 37 7 39 3 37 5 36 6 36 3 26 1 26 6 256 5 38 5 39 0 32 6 26 5 26 0 26 6 40 4 39 4 39 2 Tm 784 78 4 784 79 2 79 2 79 2 78 8 788 79 8 798 2 79 2 79 2 788 79 2 798 2 79 2 79 2 798 2 79 2 79 2 79 2 78 5 79 5 78 5 79 2 70 5 79 5 79 5 79 5 78 5 79 2 79 5 79 5 79 2 79 2 70 2 79 2 79 2 798 2 Tm 784 73 4 754 79 2 79 2 79 2 728 738 72 8 79 2 79 2 79 2 7858 79 2 792 79 2 79 2 792 79 2 792 79 2 79 5 79 5 79 5 792 79 5 79 5 79 5 795 795 79 2 Fos 79 5 79 2 79 2 79 2 792 79 2 79 2 Conc 1 OEO2 1 0E02 1 0E0 2 9E02 9 5604 4 3E02 1 2E01 1 2601 1 2E01 4 0E04 6 7E00 4 0E04 2 5E00 2 5E00 2 5E00 1 0E00 6 4E 04 476 014 5 4E 01 5 4E 04 5 4E 01 5 9E 02 9 2E 02 3 0E 02 1 8E 01 1 8E 01 1 8E 04 3 2E02 2 2E02 25602 5 0E 02 6 0E 02 6 0E 02 2 5602 3 4E02 2 2E02 2 0E 02 2 0E 02 2 0E 02 Conc 1 0E02 1 0E02 1 0E02 2 9E02 9 5604 1 3E02 1 2E04 1 2E04 1 2E04 1 0E04 6 7 E00 41 0E04 2 5E00 2 5E00 2 5E00 1 0E00 6 4E 04 4 7 E
14. c 5229TA Figure 7 Selecting the Melt Curve in the Workshop module 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 TM270 9 05 Page 9 9 Name the protocol by entering a name in the Protocol Filename box Save the protocol by selecting the Save this protocol button and then selecting Save in the Saving a Protocol window that appears Select the Run with selected plate setup button IV B Plate Setup E oe P o oe 9 6 7 8 Library 10 11 Run Time Central Select the Library module Select the View Plate Setup tab Select the Create a new plate setup button In the Edit Plate Setup window that appears select the Whole Plate loading tab instead of the Per Dye Layer mode Figure 8 Select Unknown from the sample type icons Select the wells to be used Click on the Select and load fluorophores tab Select or deselect the dyes being used from the Select and load fluorophore panel After each dye is selected assign a different color from the four colors listed Figure 9 Select a fluorophore pen and then select the wells using this fluorophore Repeat for each dye being used Enter a name in the Plate Setup Filename box Figure 8 Save the plate setup b
15. curve window amplification curve in the amplification curves window no apparent amplification above Problems with data export or instrument 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 IIL IV or V 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 II IV or V 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 1 dilute the template so that 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 Plate performance can differ from manufacturer to manufacturer Use plasticware recommended by the instrument manufacturer Instrument was
16. 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 ES 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 Part TM270 a Page 33 2 Select the Standard Curves tab to view the standard curve Figure 34 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 35 To do so select the Standard Curves tab to view the standard curve In the Edit menu select Flip Std Curve Axes See Notes 1 and 2 3 View the concentrations for all samples including the unknown samples in the table next to the standard curve graph Figures 34 and 35 The calculated concentrations can also be viewed in the sample details report Section VILF 4 Repeat Step 1 with any other desired set of standards and samples Notes 1 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 additiona
17. improperly calibrated Calibrate instrument as instructed by the manufacturer Thermal cycling conditions were suboptimal Optimize the annealing temperature Thermal cycling conditions were suboptimal 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 Part TM270 Printed in USA Page 40 210 VIII Troubleshooting continued CQ A Symptoms Causes and Comments Variability in signal among The baseline region was not set correctly The baseline should Promega replicate samples continued be flat The baseline region can be adjusted manually for each well to account for sample to sample variation Section X D The plate was not completely sealed Carefully seal the plates 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 including primer dimer Decrease the cycle number to reduce the accumulation of n
18. product See Note 6 The default melt threshold is set at 25 of the signal change for the sample within the set that has the ereatest 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 No peaks for the standard reference template or genotyping control sample indicates amplification problems See Section VIII for more information about possible causes 2 The target melt temperature range can be adjusted manually Move the mouse so 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 3 The melt threshold is the level of signal that must be reached for the Plexor Analysis Software to call the melt results Target T indicators are included in the table to the right of the amplification and melt curve windows
19. sample The concentration is entered in a pop up window following designation of a well as a standard Selecting the wells and choosing the Create Dilution Series icon can automatically create a titration curve across several wells Positive control m Color assignment Figure 29 The icons used to define samples in the Plexor Analysis Software 5048TA Figure 30 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 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
20. table in a small frame called Data Display located above the amplification curves graph 17 Left click on the cell labeled Well Cycle to select all data in the Data Display table 18 Press Control C to copy the data from the iCycler iQ software 19 Inthe Plexor Analysis Software select the blank cell shown in the amplification section of the File Import screen for the appropriate dye see Figure 18 and select the Paste From Clipboard button 20 In the iCycler iQ software select a different fluorophore pen and repeat Steps 15 19 for each dye used 5 ode Backq oinal erected 1024 08 1108 86 1149 64 1266 77 1040 43 1092 37 1167 15 1251 96 1022 95 1087 13 1161 95 1262 79 1034 95 1091 11 1142 29 1259 49 1036 00 1068 71 1147 34 1256 68 r Yar Y Y rw eee rat reed cee 5236TA Figure 20 The PCR Quantification tab with data displayed and all data selected 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 TM270 Printed in USA Page 20 I 03 21 To export the melt data go to the Melt Curve tab of the Data Analysis C module in the iCycler iQ software Figure 21 D 22 Highlight a fluorophore pen from the Select a Fluorophore list Promega 23 Once the melt curve graph is sho
21. temperature the target formed in the first round is amplified Figure 12 shows the final thermal cycling program Table 3 Thermal Cycling Profile for Genotyping Assays Number of Experimental plate well factor Automatically inserted into program collection Initial 1 extension 40 cycles extension 60 C initially 8 seconds per increasing in fluorophore 0 5 C i e 16s for 2 increments dyes Melt curve 70 cycles 1This denaturation step should be extended to 2 minutes if an external well factor plate is being used for the initial well factor collection step See Section IX G for more information 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 TM270 Printed in USA Page 12 9 05 Note The Bio Rad iCycler iQ performs a well factor collection process at the C onset of a run to normalize signal across a plate Before starting a run before cycling A begins the iCycler IQ will require designation of the experimental plate internal or an external well factor plate as the source of this data Use of the experimental plate the simplest and recommended method requires that the selected wells all contain the same concentrations of dyes If this requirement is not met an external well factor plate must be used See Section IX G and the user manual supplied with the iCy
22. the PCR Curves tab The default setting for the expected target melt temperature is 90 0 and the default target Tn range is 1 C 2 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 32 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 3 Inthe 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 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 TM270 Page 30 Printed in USA 9 05 Duplex l aan Not for Medical D PCR Curves Sample 10s tsadard Cune Reports FAM Target4 10E Target 2 Trae 4 4 1 4 1 4 i o RFU 2 5E6 Graph Legend showing Sample T and Target T indicators lt i Melt Threshold d RFUYT 2684 5051TA Figure 32 The expected target melt temperature is displayed in a table to the right of the graph 4 Optional The melt threshold may be reset to change the sensitivity in detecting the amplification
23. this button See Section VIII D i 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 X C IXF 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 100 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 increases 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 IX G Use of an External Well Factor Plate Materials to Be Supplied By the User e Bio Rad 10X Well Factor Solution The Bio Rad iCycler iQ performs a well factor collectio
24. to pipet accurately Do not pipet volumes lt 1 dilute the template so that 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 DNA by vortexing or pipetting Adjust the baseline region The baseline region can be manually adjusted for each reaction See Section X 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 lt 1 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 Plexor One Step qRT PCR System Technical Manual TM263 or Plexor Two Step qRT PCR System Technical Manual TM264 The plate was not completely sealed
25. up window This change will alter the scale for the entire data set VILD 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 IX D for information on display and adjustment of baseline regions The amplification threshold is used to determine the C value for the samples Figure 33 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 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 TM270 Page 32 Printed in USA 9 05 Duplex 1 aan Not for Medical Diagnostic Use k FCR Curves Sample ibs Standard Cures Reports A FAM Target 1 JOE Target Amplification Curves Promega Amplification Threshold 5052TA Cycle Figure 33 The amplification and melt thresholds 1 To adjust the amplification th
26. 014 5 4E 01 5 4E 04 5 4E 01 5 0E 02 9 2E 02 3 0E 02 1 8E 01 1 8E 01 1 8E 04 3 2E02 2 2E02 2 5E02 6 0E 02 6 0E 02 6 0E 02 2 5E02 3 4E02 2 2E02 2 0E 02 2 0E 02 2 0E 02 Tm eee ee ee aa a Tm a a a 2 a a a f a f a f a a G a G a i a a Ow f a ee ee Oe ee ee l oua iIa l Ma 5053TA 5054TA A 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 9 05 Part TM270 Page 35 Promega VILF Reports The Plexor Analysis Software includes 5 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 Tm and the number of melt curves that cross the melt threshold line Figure 36 A sample with a C value of N A h
27. 37 The Thresholds tab Bale 5055TA 5056TA 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 TM270 Page 36 Printed in USA 9 05 Baseline Regions The baseline regions report includes the numerical values for the C cycle number used in each sample Figure 38 o Run Info The run info report includes the information from the data import Promega Figure 39 Import Files The Import Files report includes information on the data import files Figure 40 5057TA BioRad iCycler iQ October 3 2005 5508TA Figure 40 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 TM270 fe Page 37 VILG 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 A Run Tem
28. 7 Enter information specific to your experiment in the Run Info screen Step 2 Figure 17 Other details date notes title name of the person performing the experiment etc can also be entered in the provided windows 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 TM270 9 05 Page 17 Assay Setup ma os In order to define the assay you wish to import please specify the following parameters Required _ _ _ _ _ _ _ _ Ce ere e MM Please select the supported instrument for this ioRad iCycler iQ oRad iCycler iQ via paste epheid SmartCycler ratagene Mx3000P hix3O05P Research Opticon 2 _ Cancel 5232TA Figure 16 The Assay Setup screen Run Info Please fill in the details below regarding your run Details Assay Name Instrument BioRad iCycler iQ via paste ExpeimentTile Operator tame sss s sSSS ee 5233TA Figure 17 The Run Info screen 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 TM270 Printed in USA Page 18 ae ile Import Promega To import your iCycler run copy the Data Table for each dye from the amplification and melt stages and paste them into the tables as labele
29. 71 is available for download at www promega com plexorresources The software is also available free of charge on CD ROM Software installation instructions are given in Section IX B Note Two sets of data one for the amplification data and one for the melt data must be copied from the iCycler iQ and pasted into the Plexor Analysis Software This must be repeated for each dye used 1 Launch the Plexor Analysis Software go to the Start menu and select Programs then Plexor then 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 2 Inthe File menu select Import New Run or select the icon 3 Optional Enter an assay name in the Assay Setup screen Step 1 Figure 16 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 Bio Rad iCycler iQ via paste as the instrument type 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 Note For frequently run assays a template with the target information and dyes can be saved Section IX C 6 Select Next
30. Madison WI 53711 or EraGen Biosciences Corporate Licensing 918 Deming Way Suite 201 Madison WI 53717 Phone 608 662 9000 Fax 608 662 9003 2005 Promega Corporation All rights reserved RNasin is a registered trademark of Promega Corporation ImProm II and Plexor are trademarks of Promega Corporation iCycler iQ is a trademark of Bio Rad Laboratories Inc Macintosh is a registered trademark of Apple Computer Inc Microsoft Excel 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
31. Run O Prolonged exposure of the reactions to high temperatures berfore thermal cycling may adversely affect the final results Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Begin Run Library Sample volume 25 ul Select run purpose Select well factor source PCR Quantification 2 2 Experimental Plate Melt Curve Pure Dye Calibratic Well Factor Plate Running plate setup contains no wells with the Pure Dye sample type Run Time Central 5228TA Figure 5 The Run Prep 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 TM270 9 05 Page 7 O IV Instrument Setup and Thermal Cycling for One Step qRT PCR y pP These instructions describe instrument setup and thermal cycling conditions for mega cDNA quantitation using the Plexor one step qRT PCR System The thermal cycling program includes the initial incubation for the reverse transcription IV A Thermal Cycling Program The one step qRT PCR thermal cycling program is shown in Table 2 Primers designed using the Plexor Primer Design Software have an annealing temperature of approximately 60 C Figure 7 shows the final thermal cycling program Note The Bio Rad iCycler iQ performs a well factor collection process at the onset of a run to normalize signal across a plate Dur
32. Software To launch the Plexor Analysis Software go to the Start menu and select Programs then Plexor then 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 In the File menu select Import New Run or select the icon Ma Optional Enter an assay name in the Assay Setup screen Step 1 Figure 25 This screen is used to enter general information about the type and format of the data that will be used for each assay 5237TA Figure 24 The Melt Curve tab with data displayed and all data selected 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 TM270 Page 24 Printed in USA 9 05 Assay Setup rp Ez In order to define the assay you wish to import please specify the following parameters Required Assay Name Please enter the name of this assay Instrument Please select the supported instrument for this assay oche LightCycler2 ioRad iCycler iQ loRad iCyeler iQ via paste epheid SmartCycler Cancel
33. Technical Manual Plexor Systems Instrument Setup and Data Analysis for the Bio Rad iCycler iQ Real Time PCR Detection System INSTRUCTIONS FOR USE OF PRODUCTS A4011 A4021 A4031 A4041 A4051 AND A4061 PRINTED IN USA 9 05 Part TM270 Plexor Systems Instrument Setup and Data Analysis Vii for the Bio Rad iCycler iQ Real Time PCR Detection System Di ON ce soccer N E AT 2 II Plate Preparation and Amplification esseesesesseessesesssessesesesneesesseeneeneesneeseens 2 III Instrument Setup and Thermal Cycling for qPCR and Two Step qRT PCR 3 Pi Pineal Cy lin PO elisa grape eovonnieae EE sacar meneame aninness 3 Bo csi 06 c NNT nT PP Enon errr TENT Tr eer te ee eee es 5 IV Instrument Setup and Thermal Cycling for One Step qRT PCR eeeeeeeeees 8 A Thermal Cycling Program se sssssssssseereeresssssssreteereeesssssrttreeereesssssrrrreenssssssrrrrreeenenensneens 8 Eo Tae ea a EE 10 V Instrument Setup and Thermal Cycling for Genotyping SNP Assays 12 As 0s a gic Fe ycline PrOCTAI sirni oiiire ATAR 12 B PS E EA E A E E ecaeeececeeee 14 VI Data Export from the iCycler iQ and Import into the Plexor Analysis Software 0 ccessesssessssesnecsecsseeseeseesneenneennsen 17 A Pasting Data Directly into the Plexor Software sssssseessssssssssssssssrrrrrrreeesreeese 17 Bi Data Exportand Import via EXCEl ass ccetetasasscdesinnoisas vu oaaieuceaconatn
34. al 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 appears abnormal in Be sure that the display settings for the computer are set to the Plexor Analysis Software 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 is 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 is too low Optimize the annealing temperature Reactions were assembled incorrectly Verify that the appropriate primer pairs were added Genotyping Miscalled known
35. as an amplification curve that did not cross the amplification threshold PCR Cumas Sample IDs Standard Cues Reports Sample Detaits Thresholds Baseline Regions Run Into Import Fites ati Sample ID JOE Type 1 Cone Exp Tm Tm A Standard 100 Standard 10602 Yes 1 m2 Standard 100 Standard 1 0602 Yeo 1 Standard 100 Sample 1 5 Sample 2 Standard F r 1 0602 Yes Yes A Sample 3 Standard 124 Standard 125 HS RRBs 2 8 8 88 Standard 0 54 E Standard Standard 0 54 Standard r f A Standard Sample 10 Unknown 7 BE Unikn ovun Sample 11 Jnkr 7 7 5 E 02 Unikn own Sample 12 Unknown ji 0E 02 Unikn own S amp F 38 Figure 36 The Sample Details tab Thresholds The thresholds report includes the numerical values for the thresholds in the current analysis Figure 37 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 IX C for more information about creating an analysis template PCR Cunes Sample IDs Standard Curves Reports Sample Details Thresholds Baseline Regions Run Info import Files Blas Amp Threshold Curent Amp Threshold Standard Deviations Meli Call Threshold Current Melt Threshold Percentage Expected Tm Lower Threshold Expected Tm Expected Tm Upper Threshold Figure
36. ate Setup View Quantities Identifiers Run Prep va cles Save this protocol Promega Run with selected plate setup Protocol Filename Plexor amp tmo Selected Plate Setup Template pts All changes must be saved before running Selected Plate Setup is loaded in the Edit Plate Setup tab Show options Select data collection step s Infinite Hold Increment Time Cycle 1 Gradient Decrement Time aiMelt Curve C Ramping O Step 1 _ Increment Temperature Cycle Description Run Time P JCF E Cycle 2 Decrement Temperature Step Process Central O Step 1 lt x Fast Step 2 Cycle 3 WI Step 1 cle Repeats e a SS tear ee 8 seconds required per dye i e time must be increased if multiple dyes are used La 5225TA Figure 2 The Selecting the Melt curve in the Workshop module ILB Plate Setup 1 Select the Library module 2 Select the View Plate Setup tab 3 Select the Create a new plate setup button 4 Inthe Edit Plate Setup window that appears select the Whole Plate loading tab instead of the Per Dye Layer mode Figure 3 Select Unknown from the sample type icons Select the wells to be used Click on the Select and load fluorophores tab 2 N A Select or deselect the dyes being used from the Select and load fluorophore panel After each dye is selected assign a different color from the fou
37. ature T m 65 0 95 0 C a0 0 Target Tm Upper Bound 0 0 5 0 C fi Oo eae PO ENS pee m m m n LT ene ay 5061TA Figure 42 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 Tn can be determined A Tm 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 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 Tm 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
38. cells by double clicking on the desired cells Repeat Dwell Time Setpoint Melt Curve and Temp The Dwell Time for the final melt step must be 00 08s per dye used Figure 7 bottom panel Note The repeat number for the final melt step must be entered before the Melt Curve box can be checked 7 Inthe Select data collection step s panel select Cycle 3 Step 2 and Cycle 4 Step 1 for data collection if not already indicated by double clicking the desired Step a camera icon indicates that a step is selected for data collection Figure 7 Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Save this protocol Run with selected plate setup Protocol Filename Plexor 1 step RT tmo Selected Plate Setup Template pts All changes must be saved before running Selected Plate Setup is loaded in the Edit Plate Setup tab Show options Select data collection step s _ Infinite Hold Increment Time Cycle 2 a Gradient Decrement Time J aiMelt Curve Cl Ramping O Step 1 Increment Temperature Cycle Description Run Time r 5 i a Cycle 3 Decrement Temperature Step Process Central Oo Step 1 lt W Step 2 Cycle 4 lt I Fest Step 1 Insert Cycle Delete Cycle Insert Step Delete Step 8 seconds required per dye i e time must be increased if multiple dyes are used Data Analysis a a
39. cler iQ instrument for more information on use of an external well factor plate Promega 1 Launch the iCycler iQ software Select the Library module Select the View Protocol tab Figure 11 Select the Create a new protocol button a e fe N The Workshop module will appear Select the Melt Curve checkbox from the Show options panel Figure 12 6 Create the protocol shown in Figure 12 as follows e Use the Insert Cycle Delete Cycle Insert Step and Delete Step buttons Figure 12 to create a new protocol To insert or delete a cycle or step select the appropriate button then select the position of the cycle step in the protocol table Deselect the active button before directly editing the cells e Directly edit cells by double clicking on the desired cells Repeat Dwell Time Setpoint Melt Curve and Temp The Dwell Time for the final melt step must be 00 08s per dye used Note The repeat number for the final melt step must be entered before the Melt Curve box can be checked O If an external well factor plate is used the initial denaturation time must be extended to 2 minutes 7 Inthe Select data collection step s panel select Cycle 2 Step 2 and Cycle 3 Step 1 for data collection if not already indicated by double clicking the desired Step a camera icon indicates that a step is sel
40. 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 TM270 9 05 Page 45 Run Template Ea Ez Set the sample types colors target concentrations and sample ID s for each sample using the well selector below Plate Setup Sample tds r Assign sample types colors and concentrations a e O st IV Propagate Selection Across Dyes FAM Target 1 JOE Target I LON MO oe Oe lt 9 N A o o N A o o N A o oO oi oa m j m I 5060TA Figure 41 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 42 Note Descriptions of the analysis details are provided below 3 Select Exp
41. ctors in the assumed internal well factor data collection that is automatically inserted by the iCycler iQ software This inserted function is a 90 second process at denaturing temperatures Setup of an External Well Factor Plate 1 Dilute the 10X External Well Factor Solution to 1X in ddH O Twenty five microliters of this 1X solution are required for each well used in the experimental plate with additional volume prepared for pipetting variation Dilute 300ul of 10X External Well Factor Solution in 2 7ml water for one plate 2 Aliquot 25ul volume equal to the amplification reactions to an empty plate Well positions not used in the plate setup process II B IV B and V B do not need this solution 3 Seal plate and centrifuge briefly 4 Inthe Run Prep window that opens enter 25 for Reaction Volume and select the Well Factor Plate radio button for the Select well factor source option Place the Well factor plate into the instrument and immediately begin the run by pressing the Begin Run button Keep the experimental plate on ice 5 After several minutes you will be promted to remove the well factor plate and insert the experimental plate Select continue when the experimental platehas been placed into the instrument and the lid has been closed Further details on use of a well factor plate are available in the iCycler iQ instrument manual IX H Reference 1 Bustin S A 2004 A Z
42. d below Amplification F JOE Zn cancel lt Back Finish 5234TA Figure 18 The File Import screen 8 Select Next Use the File Import screen Step 3 Figure 18 to paste data exported copied from the iCycler iQ as described in Steps 9 23 below 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 9 In the iCycler iQ software select the Library module 10 Select the View Post Run Data tab Figure 19 11 Highlight the data run file opd from the Data Files list ot Jarrds 1160p 5235TA Figure 19 The View Post Run Data tab in the Library module 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 TM270 fe Page 19 CQ 12 Select the Analyze Data button v 13 Within the Data Analysis module that opens select the PCR Promega Quantification tab 14 Select Background Subtracted from the Select analysis mode drop down menu Figure 20 15 Highlight a fluorophore pen from the Select a Fluorophore list 16 Right click on the amplification curves graph and choose Display Data This will display a
43. e is being used for the initial well factor collection step See Section IX G for more information 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 TM270 9 05 Page 3 View Protocol View Plate Setup View Quantities Identifiers View Post Run Data ac Workshop Program Files Bio Rad S iCycler New Folder Viewing Protocol 2Step tmo Selected Plate Setup Template pts Protocol Files Edit this protocol 25tep tmo Create a new protocol Run with selected plate setup genotyping_external well fact_O_8s ramp tmo E Report Files ing i gt Rep genotvprig int well fact _0_8s ramp tmo Print this protocol Selected Plate Setup may be changed in the View Plate Setup tab 5224TA Figure 1 The View Protocol tab in the Library module a ee N e 7 8 9 Launch the iCycler iQ software Select the Library module Select the View Protocol tab Figure 1 Select Create a new protocol The Workshop module will appear Select the Melt Curve checkbox from the Show options panel Figure 2 Create the protocol shown in Figure 2 as follows e Use the Insert Cycle Delete Cycle Insert Step and Delete Step buttons Figure 2 to create a new protocol To insert or delete a c
44. e the synthesis of primer dimer or nonspecific product Check for signal bleedthrough Calibrate the instrument as instructed by the manufacturer 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 Altenatively 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 Part TM270 a Page 39 CO VIII Troubleshooting continued a Symptoms Causes and Comments Promega Broad melt curve or a shoulder on 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 by the manufacturer for the dye set used Decrease the primer concentration e g 0 1uM Be sure the thermal cycler is programmed correctly Section IIL IV or V No melt curve observed in the Poor amplification See causes and comments for Flat melt
45. e 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 Part TM270 Page 50 Printed in USA 9 05 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 A curve and generate the new standard curve using the samples you have selected You may generate the original standard curve at any time Promega 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 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
46. ecrement Temperature Step Process Central gO Step 1 amp gt Fast Step 2 Cycle 4 Fest Step 1 n Insert Cycle Delete Cycle Insert Step Delete Step v 8 seconds required per dye i e time must be increased if multiple dyes are used 5231TA Figure 12 The selecting the Melt Curve in the Workshop module 8 Name the protocol by entering a name in the Protocol Filename box Save the protocol by selecting the Save this protocol button and then selecting the Save button in the Saving a Protocol window that appears 9 Select the Run with selected plate setup button V B Plate Setup 1 Select the Library module 2 Select the View Plate Setup tab 3 Select the Create a new plate setup button 4 Inthe Edit Plate Setup window that appears select the Whole Plate loading tab instead of the Per Dye Layer mode Select Unknown from the sample type icons Figure 13 Select the wells to be used Click on the Select and load fluorophores tab 2 N A Select or deselect the dyes being used from the Select and load fluorophore panel After each dye is selected assign a different color from the four colors listed Figure 14 9 Select a fluorophore pen and then select the wells using this fluorophore Repeat for each dye being used If all of the wells in use do not have the same dyes designated an external well factor
47. ected for data collection Figure 12 View Protocol View Plate Setup View Quantities Identifiers View Post Run Data fe Protocol Files c Edit this protocol aac p Program Files Create a new protocol Run with selected plate setup Bio Rad S iCycler New Folder genotyping_external well Fact_O_8s ramp tmo DReport Files i R p Rep joractyaing i well Fact_O_8s ramp tmo priie thas ma Viewing Protocol 2Step tmo Selected Plate Setup Template pts Selected Plate Setup may be changed in the View Plate Setup tab Workshop 5224TA Figure 11 The View Protocol tab in the Library module 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 TM270 9 05 Page 13 Edit Protocol Show options _ Infinite Hold Gradient Edit Plate Setup _ Increment Time Decrement Time View Quantities Identifiers Run Prep Save this protocol Run with selected plate setup Protocol Filename Plexor Genotyping tmo Selected Plate Setup Template pts All changes must be saved before running Selected Plate Setup is loaded in the Edit Plate Setup tab Select data collection step s Cycle 2 A MjMelt Curve Cl Ramping O Step 1 5 _ Increment Temperature Cycle Description Run Time P LCY Pp Cycle 3 D
48. ection VI IMI A Thermal Cycling Program The thermal cycling program for qPCR and two step qRT PCR is shown in Table 1 Primers designed using the Plexor Primer Design Software have an annealing temperature of approximately 60 C Figure 2 shows the final thermal cycling program Note The Bio Rad iCycler iQ performs a well factor collection process at the onset of a run to normalize signal across a plate Before starting a run before cycling begins the iCycler iQ will require designation of the experimental plate internal or an external well factor plate as the source of this data Use of the experimental plate the simplest and recommended method requires that the selected wells all contain the same concentrations of dyes If this requirement is not met an external well factor plate must be used See Section IX G and the user manual supplied with the iCycler iQ instrument for more information on use of an external well factor plate Table 1 qPCR and Two Step RT PCR Thermal Cycling Program Number of Experimental plate well factor Automatically inserted into program collection Initial 1 D 40 cycles LAT a a d 8 econdesef per 60 C initially fluorophore increasing in ie 8 16 24 Melt curve 0 5 C or 32s for 1 2 70 cycles increments 3 or 4 dyes respectively 1This denaturation step should be extended to 2 minutes if an external well factor plat
49. entrifuging a 96 well plate e optical quality sealing tapes e Bio Rad 10X External Well Factor Solution 1 After the amplification reactions are assembled cover the reaction plate with an optical quality sealing tape Centrifuge briefly to collect the contents at the bottom of each well Note Keep the plate on ice during reaction setup and programming of thermal cycling conditions 2 Program the Bio Rad iCycler iQ Real Time PCR Detection System The proper thermal cycling conditions and instructions for programming the instrument are provided in Section III qPCR and two step qRT PCR assays Section IV one step qRT PCR assays and Section V 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 TM270 Page 2 Printed in USA 9 05 III Instrument Setup and Thermal Cycling for qPCR and Two Step qRT PCR CQ A These instructions describe instrument setup and thermal cycling conditions for DNA or cDNA quantitation using the Plexor qPCR or Plexor Two Step qRT Pi omega PCR Systems Thermal cycling programs described in this manual are optimized to work with primers designed using the Plexor Primer Design Software which can be accessed at www promega com plexorresources Instructions for data export from the Bio Rad iCycler IQ into the Plexor Analysis Software are provided in S
50. es icon 3 Select the samples to be adjusted using the well selector Note The baseline region can be adjusted for individual samples or eroups 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 43 For multiplex assays the baseline is set independently for each dye 4 Adjust the upper limit of the baseline region for each sample so the upper limit is approximately 5 cycles 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 5 If necessary adjust the lower limit to a region that creates the flattest baseline given the selected upper limit 6 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 VII D See Notes 3 and 4 PCR Cumes Sample IDs t2r 8 14 Reports Baseline FAM Target 1 Joe Target 2 re g 0 N Well Semple l0 ct Tm Conc Tm Tm Bi Sample5 314 792 Yes 4 RFU 25E5 Lower limit Upper limit Figure 43 An am
51. gin Run Sample volume 25 ul Select run purpose Select well factor source gt PCR Quantification G Malt Curve Experimental Plate Pure Dye Calibration Well Factor Plate R ossessecosesossosoeseseecoscocesosoesosocosseosesoseaosse Running plate setup contains no wells with the Pure Dye sample type Run Time Central 5230TA Figure 10 The Run Prep 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 TM270 9 05 Page 11 C V Instrument Setup and Thermal Cycling for Genotyping SNP Assays 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 V A Thermal Cycling Program The thermal cycling program is shown in Table 3 Each of the genotyping primers includes bases that are not complementary to the target sequence at the 5 end The annealing temperature for the first round of amplification is 50 C to allow the primer to anneal and be extended Subsequent rounds are performed using an annealing temperature of 60 C which is the melting temperature Tn of the primer pair including the noncomplementary bases During subsequent rounds of amplification performed at the higher annealing
52. gure 20 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 wells 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 well 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 wells within a dilution series of standards may be assigned as standards simultaneously by highlighting multiple wells and using the Create Dilution Series function 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 cycl
53. ing the final steps of starting a run before cycling begins the iCycler iQ will require designation of the experimental plate internal or an external well factor plate as the source of this data collection The high temperature of this process is not compatible with the reverse transcriptase enzyme therefore an external well factor plate and subsequent selection must be analyzed before the experimental plate is inserted into the iCycler iQ instrument See Section IX G and the user manual supplied with the iCycler IQ instrument for more information on use of an external well factor plate 1 Launch the iCycler iQ software 2 Select the Library module 3 Select the View Protocol tab Figure 6 Table 2 One Step qRT PCR Thermal Cycling Program f Step Temperature Time umbene Cycles Reverse transcription reaction Reverse transcription reaction reaction 5 minutes p teyde teyde SS E E Initial denaturation and inactivation of the ImProm I 95 C 2 minutes 1 cycle Reverse Transcriptase 40 cycles Annealing and extension 8 seconds per 60 C fluorophore initially i e 8 16 24 Melt curve increasing in or 32s for 1 70 cycles 0 5 C 2 3 0r 4 increments dyes respectively The length of incubation for the reverse transcription reaction can be increased to up to 30 minutes Longer incubation times can lead to increased sensitivity but also higher background Promega Corporati
54. l 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 2 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 a Standard Curve The Remove a 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 TM270 Page 34 Printed in USA 9 05 PCR Curves Sample IDs Standard Curves Reports FAM Target 4 Curve 1 Standard Curwe 1 y 0 30x 10 44 Well Ad Sample ID Standard 100 Standard 100 Standard 100 Log Concentration 4 5 R 0 993 CE H3 NTC does not have a valid Ct value F3 H2 NTC does not have a valid Ct value F4 lt gt H1 NTC does not have a valid Ct value FS Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp Stand Stand Stand Samp Samp Samp ed e2 e3 ard 12 5 ard 12 5 ard 12 5 e4 a5 eG ad 25 ard 2 5 ard 2 5 eT e8 eg
55. lect a fluorophore 2 Assign a color 3 Enter plate notes C 3 530 ROX 575 C 5 635 SYBR 490 M FAM 490 TAMRA 530 AS Color not used HEX 530 I TAMRA 548 AS mj J0E 530 TET 490 J0E 530 LC640 635 C TET 530 Color not used lt j gt A Erase Available Filter Wheels FilterSet4 Define sample wells 4 Load well fluorophores by clicking to select a Huor pen and then clicking wells Dooce AAA 5227TA Figure 9 The Select and load fluorophores tab in the Edit Plate Setup window 12 The Run Prep window will open Figure 10 Enter a sample volume of 25ul and select the Well Factor Plate radio button under the Select well factor source option 13 Create an external well factor plate as described in Section IX G 14 Seal the external well factor plate and centrifuge briefly 15 Place the well factor plate into the instrument and immediately begin the run by pressing the Begin Run button Keep the experimental plate on ice O Prolonged exposure of the reactions to high temperatures berfore thermal cycling may adversely affect the final results 16 After several minutes you will be prompted to remove the well factor plate and insert the experimental plate Select continue when the experimental plate has been placed in the instrument and the lid has been closed Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Be
56. lows you to assign the sample type Unknown to all selected samples Select one or more wells 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 gt 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 wells 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 the concentration 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 wells 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 a
57. n process at the onset of a run to normalize signal across a plate During the final steps before starting a run before cycling begins the iCycler IQ will require designation of the experimental plate or an external well factor plate as the source of this data Use of the experimental plate the simplest and recommended method requires that the selected wells all contain the same concentrations of dyes If not all wells share the same dyes and dye concentrations an external well factor plate must be used For example if the Plexor System positive control reaction FAM labeled primer only were to be run on the same plate as a multiplex assay an external well factor plate would be required 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 TM270 9 05 Page 51 Bio Rad supplies a 10X External Well Factor Solution to be used for creation of an external well factor plate The process of creating an external well factor plate is described below Note If an external well factor plate is being used for qPCR or Two Step RT PCR Section III or Genotyping SNP detection assays Section V the thermal cycling protocol must be modified to include a 2 00 minute initial denaturation The thermal cycling protocols described in Sections III and V of this technical manual include a 30 second initial denaturation that fa
58. nappropriate 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 Increasing fluorescence over time Excessive template was added to the reactions Dilute the template and repeat the experiment 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 IX D The baseline region was set too close to the signal change Manually adjust the baseline region Section X D Two or more distinct melt curves in For the Plexor qRT PCR Systems both RNA and DNA the melt curves window templates can be amplified Treat the RNA template with RNase free 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 non target sequences Optimize the annealing temperature Increase the annealing temperature by increments of 2 C to reduce the synthesis of primer dimer 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 dimer or nonspecific product Reduce the number of amplification cycles to minimiz
59. nd 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 Part TM270 9 05 Page 49 Oo Lng BR ooo mmm Neo Assign Standard shortcut r The Assign Standard function allows you to assign the sample type standard to all selected samples Select one or more wells 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 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 Create Dilution Series shortcut f The Create Dilution Series function creates a full dilution series within a row or column of wells Select the wells 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 Fi
60. ntnanrsonvawascatea tenes 22 VII Data Analysis with the Plexor Analysis Software cscsessseseseceeseeseeseeneeneeneense 28 A 20h 9 Demit a Werpererer verrreeseter ert ceeererrr tree reeertnrtntrtrerr rrr rreetrr vir cerieevre rir rei srrerraresweerr errr 28 B Adjusting the Expected Target Melt Temperature oc eesesesesseseseeseessesneenes 30 C Adjusting the Y Axes of the Amplification and Thermal Melt Carves OPUONAl essiccare 32 D Adjusting the Baseline Region and Amplification Threshold Line Optional 32 E Generating a Standard Curve Optional sssssessesssssssssssrerrreessssssrrrrrereesssssrrrreeesrssssss 33 PREO a E EAA EEEE AE E 36 G Saving and Printing the Analysis File sssseeeeessssssssseeersresssssssrrrressrssssssrrrrressesnsseens 38 MU Troupleshootiio aiino AEE e EAE EEE 38 IX APENI ordii eee 45 A Plexor Analysis Software Operating System Compatibility oes 45 By Plexor Analysis Software Installation svamaunwisesscaminsaisesevtsseusion mums 45 C Advanced Options ocala ctdcctn npc vent ieeosesetst0 tia caceeee eee ore nee 46 D Manual Baseline Adj stMEN Sieisen eisini ee 48 E Pon S on E NTT E E 49 F Amplification Efficiency Calculations ssssssssssssssssseeessreeereeesssssssssssssssssrrrrreeeeesreesss 51 Se seer a External yell Factor Te Ate siseses 51 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 ww
61. 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 Part TM270 Page 52 Printed in USA 9 05 The PCR process which is the subject of European Pat Nos 201 184 and 200 362 owned by Hoffmann LaRoche is covered by patents issued and applicable in certain countries Promega does not encourage or support the unauthorized or unlicensed use of the PCR process Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license The above primary European Pat Nos 201 184 and 200 362 will expire 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 non exclusive non transferable 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 uses please contact Promega Corporation Licensing 2800 Woods Hollow Road
62. oftware be sure to choose the amplification and melt curve files generated using the same data The file names assigned when exporting data must be descriptive so 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 IX 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 TM270 Printed in USA Page 26 a03 To import your iCycler run use the file dialogs below to specify the files for each dye used for amplification and aie poles below Promega Cancel lt Bac Finish 5240TA Figure 27 The File Import screen 33 Select Finish to complete the data import and to open Analysis 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 Part TM270 as Page 27 VII Data Analysis with the Plexor Analysis Software After data import is complete Section VII the PCR Curves tab of the Analysis Desktop is displayed Figure 28 File Edit View Window Help a a a B E ele olm xfs BIR Duplex
63. on 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 TM270 Printed in USA Page 8 ae View Protocol View Plate Setup View Quantities Identifiers View Post Run Data o Sc o amp ocol Files a Edit this protocol m Ici 25Step tmo tala Somat ata Create a new protocol Bio Rad 35tep tmo dynamicwF tmo SViCycler bernalat externalwF tmo Run with selected plate setup genotyping tmo E New Folder genotyping_external well fact _0_8s ramp tmo Report Files ing i 2 y Rep genotyping dnt well fact _0_8s ramp tmo Print this protocol Viewing Protocol 2Step tmo Selected Plate Setup Template pts Selected Plate Setup may be changed in the View Plate Setup tab Workshop 5224TA Figure 6 The View Protocol tab in the Library module 4 Select the Create a new protocol button 5 The Workshop module will appear Select the Melt Curve checkbox from the Show options panel Figure 7 6 Create the protocol shown in Figure 7 as follows e Use the Insert Cycle Delete Cycle Insert Step and Delete Step buttons Figure 7 to create a new protocol To insert or delete a cycle or step select the appropriate button then select the position of the cycle step in the protocol table Deselect the active button before directly editing the cells e Directly edit
64. on 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 The scale of the Y axis of the amplification curve is 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 VII 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 Part TM270 9 05 Page 41 VIII Troubleshooting continued Symptoms Nonlinear standard curve low R values Causes and Comments An amplification inhibitor is 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
65. onspecific amplification products Primers designed with another primer design software or for another amplification 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 new reagents and solutions Take precautions to prevent contamination see the Plexor gPCR 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 by the manufacturer Vertical fluorescence spikes or Consult the instrument manufacturer s user s guide 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 plate Poor amplification can lead to improper data scaling making 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 by the manufacturer Small signal change in No amplification or poor amplification See causes and amplification curve and melt comments for Flat amplificati
66. ort 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 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 VILD 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 TM270 Page 46 Printed in USA 9 05 Analysis Defaults Set the default parameters for analyzing the targets in this assay Promega Analysis Details FAM Target 1 JOE Target 2 Default Amp Threshold RFU Baseline Noise Standard Deviations foo lt sSSCi Default Melt Threshold dfRFUYdT Percentage 0 0 100 0 25 0 Expected Target Melt Temper
67. plate and Analysis Template from an existing analysis can be exported and used in future analyses Section IX C VIII Troubleshooting Symptoms Flat amplification curve in the amplification curves window no apparent amplification Causes and Comments 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 is 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 Be sure that the reactions were assembled correctly see the Technical Manual supplied with the Plexor System Thermal cycler was programmed incorrectly Verify cycle times and temperatures Section III IV or V 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
68. plate must be used See Section IX G for more information 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 TM270 Page 14 Printed in USA 9 05 10 Enter a name in the Plate Setup Filename box Figure 13 Save the plate C setup by selecting Save this plate setup and then selecting Save in the D Saving a Plate Setup window that appears Promega 11 Select Run with selected protocol 12 The Run Prep window will open Figure 15 Enter a sample volume of 25ul and check that the Experimental Plate radio button is selected under the Select well factor source option unless you are using an external well factor plate as described in Section IX G 13 Place the PCR plate into the instrument and immediately begin thermal cycling by selecting Begin Run Prolonged exposure of the reactions to high temperatures before thermal cycling may adversely affect the final results Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Plate Setup Filename Custom pts Selected Protocol Plexor amp tmo Save this plate setup All changes must be saved before running Selected Protocol is loaded in the Edit Protocol tab E AA OF Samples Whole Plate loading Select and load fluorophores clicka sample icon for loading wells Click Cursor to
69. plification window showing the baseline region and baseline upper and lower limits 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 TM270 Printed in USA Page 48 210 5510TA Notes C 1 amp 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 4 To reset 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 IXE 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 wells 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 al
70. r Analysis Software Cat A4071 is available for download at Promega www promega conyplexorresources It is also available free of charge on CD ROM by request Software installation instructions are given in Section IX B Note Two files one for the amplification data and one for the melt data must be exported for each dye used in the run 1 ae oS Data 01 Jan 05 1116 0pd Data 07 Jan 05 1700 Data 11 Jan 05 1314 0pd Data 15 Dec 04 0910 o0pd Data 15 Dec 04 0910 2 0pd Feb 05 190 i Vata 1 i J opg Data 19 Feb 05 0929 0pd Data 19 Feb 05 1129 0pd Data 19 Feb 05 1359 0pd Data 19 Feb 05 1737 opd Data 20 Feb 05 1015 opd Data 22 Feb 05 1802 opd In the iCycler iQ software select the Library module Select the View Post Run Data tab Figure 22 Highlight the data run file opd from the Data Files list Select the Analyze Data button Within the Data Analysis module that opens select the PCR Quantification tab Select Background Subtracted from the Select analysis mode drop down menu Figure 23 Highlight a fluorophore pen from the Select a Fluorophore list Right click on the amplification curves graph and choose Display Data This will display a table in a small frame called Data Display located above the amplification curves graph Left click on the cell labeled Well Cycle to select all data in the Data Display
71. r colors listed Figure 4 9 Select a fluorophore pen and then select the wells using this fluorophore Repeat for each dye being used O If all of the wells in use do not have the same dyes designated an external well factor plate must be used See Section IX G for more information 10 Enter a name in the Plate Setup Filename box Figure 3 Save the plate setup by selecting Save this plate setup and then selecting Save in the Saving a Plate Setup window that appears 11 Select Run with selected protocol 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 TM270 9 05 Page 5 Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Plate Setup Filename Custom pts Selected Protocol Plexor amp tmo Save this plate setup All changes must be saved before running Selected Protocol is loaded in the Edit Protocol tab eee ner a eee l rome ga E Samples Whole Piate loading Select and load fluorophores Click a sample icon for loading wells Click Cursor to view a well s contents Next replicate number is gt 2 e Layer Cursor Standard Unknown Blank Control Control Pure Dye Erase Per Dy Whole Plate loading All dye layers in a well must contain the same sample type and replicate Units for this plate
72. reshold based on a selected set of samples highlight the desired samples in the well selector In the Edit menu 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 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 VILE Generating a Standard Curve Optional Amplification 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 VILA For multiplex assays standard curves must be generated for
73. stalling the most recent version of the software Following installation the program can be accessed in the Start menu Programs Plexor 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 the software installation IX C Advanced Options At the File Import screen Step 3 Figure 18 Figure 27 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 41 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
74. 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 Ta 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 Part TM270 9 05 Page 47 CQ IX D Manual Baseline Adjustments y The proper baseline region is important for optimal analysis of Plexor System Promega 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 Baselin
75. the Data Display table 1024 08 1108 86 1149 64 1266 77 1040 43 1092 37 1167 15 1251 96 1022 95 1087 13 1161 95 1262 79 a 1034 95 1091 11 1142 29 1259 49 D 4 1036 00 1088 71 1147 84 1256 68 46 BARE E EEO gt aie TY AA T et ee der A M E Ps ee el E A P rd L r Pt eae iar a RERa ESE 4 s ee r PET Vi S aN Ge a din Me di Men de DESO 4 i i Ea ERTI Fives m a oO pu Q a E oOo a uw a m o ao pe Q co 5236TA Figure 23 The PCR Quantification tab with data displayed and all data selected 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 TM270 9 05 Page 23 19 20 2i 22 23 24 pia 26 d RFU dT Copy the data from the iCycler iQ software Open a new Excel spreadsheet select well A1 and paste the values into the spreadsheet Save this spreadsheet as a txt tab delimited file with a name that describes the dye and indicates that it is a melt curve data set i e Plexor FAM melt txt Select a different fluorophore pen and repeat Steps 7 12 for each dye used Transfer the txt files onto removable storage media or to an accessible network location for data transfer These files are now ready for use with the Plexor Analysis
76. tion control for the in the RNA preparation Treat the RNA template with Plexor qRT PCR Systems RNase free DNase to remove contaminating DNA Design new primers to span introns to avoid amplification of contaminating genomic DNA Assemble reactions on ice to minimize the accumulation of nonspecific amplification products including primer dimer Decrease the number of amplification cycles to reduce accumulation of nonspecific amplification products Design new primers to minimize the synthesis of nonspecific amplification products 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 Use positive displacement pipettes or aerosol resistant tips to reduce cross contamination during pipetting Use a separate work area and pipette 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 Sections III IV or V Instrument setup problems can cause amplifications to fail Consult the instrument manufacturer s user s guide for more information about potential instrument problems The Plexor Master Mix may have lost activity
77. view a well s contents Next replicate number is Cursor Standard Unknown Blank Control Control Pure Dye Erase Per Dye Layer 7 Whole Plate loading All dye layers in a well must contain the same sample type and replicate Units for this plate a Penn 2 l Define a dilution series M Scientific Notation Run Time Central lt gt Define sample A1 and replicates Standard Quantity Sample Identifier Data Analysis 5226TA Figure 13 The Whole Plate Loading tab in the Edit Plate Setup 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 TM270 9 05 Page 15 fk Samples Whole Plate loading Select and load fluorophores 1 Select or deselect a fluorophore 2 Assign a color 3 Enter plate notes Prom C1 CY3 530 ROX 575 C C 5 635 CI S BR 490 VI FAM 490 CI TAMRA 530 CI HEX 530 CI TAMRA 548 TJ JOE 530 _ TET 490 C LC640 635 C TET 530 AS Color not used lt 5 A Erase Available Filter Wheels FilterSet4 Define sample wells 4 Load well fluorophores by clicking to select a Fluor pen and then clicking wells that contain a defined sample 5227TA Figure 14 The Select and load fluorophores tab in the Edit Plate Setup window Edit
78. w promega com Printed in USA Part TM270 9 05 Page 1 II 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 Plexor qPCR System Plexor Two Step qRT PCR System and Plexor One Step qRT PCR System with the Bio Rad iCycler iQ Real Time PCR Detection System Instructions are included for instrument setup data transfer from the instrument to the Plexor Analysis Software and data analysis 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 TM2064 When using the Plexor qPCR and qRT PCR Systems 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 This ensures that the reactions will not be 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 e centrifuge capable of c
79. wn select the dF dT vs Temperature tab to display the melt derivative 24 Right click on the melt curves graph and select Display Data This will display a table in a small frame called Data Display above the melt curves graph 25 Left click on the cell labeled Well Temp to select all data in the Data Display table 26 Copy the data from the iCycler iQ software 27 Inthe Plexor Analysis Software select the blank cell shown in the amplification section of the File Import screen for the appropriate dye see Figure 18 and select the Paste From Clipboard button 28 In the iCycler iQ software select a different fluorophore pen and repeat Steps 23 27 for each dye used 29 When tables have pasted selections in the Plexor Analysis Software Figure 18 select Finish to complete data import and to open the Analysis Desktop 90 92 94 96 98 100102 Figure 21 The Melt Curve tab with data displayed and all data selected 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 TM270 9 05 Page 21 VI B Data Export and Import via Excel A The Plexo
80. y selecting Save this plate setup and then selecting Save in the Saving a Plate Setup window that appears Select Run with selected protocol All changes must be saved before running E Samptes Whole Piate loading Select and load fluorophores Click a sample icon for loading wells Click Cursor to view a well s contents Next replicate number is a Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Plate Setup Filename Custom pts Selected Protocol Plexor amp tmo Save this plate setup Selected Protocol is loaded in the Edit Protocol tab Run with selected protocol rR OOO 08 a Cursor Standard Unknown Blank Control Control Pure Dye Whole Plate loading All dye layers in a well must contain the same sample type and replicate Units for this plate a Ean Il Define a dilution series M Scientific Notation lt gt Define sample A1 and replicates Standard Quantity Sample Identifier T a n m o N w pad 5226TA Figure 8 The Whole Plate Loading tab in the Edit Plate Setup 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 Part TM270 Page 10 Printed in USA 9 05 Samples Whole Plate loading Select and load fluorophores 1 Select or dese
81. ycle or step select the appropriate button then select the position of the cycle step in the protocol table Deselect the active button before directly editing the cells e Directly edit cells by double clicking on the desired cells Repeat Dwell Time Setpoint Melt Curve and Temp The Dwell Time for the final melt step must be 00 08s per dye used Figure2 bottom panel Note The repeat number for the final melt step must be entered before the Melt Curve box can be checked If an external well factor plate is used the initial denaturation time must be extended to 2 minutes See Section IX G for further details In the Select data collection step s panel select Cycle 2 Step 2 and Cycle 3 Step 1 for data collection if not already indicated by double clicking the desired Step a camera icon indicates that a step is selected for data collection Figure 2 Name the protocol by entering a name in the Protocol Filename box Save the protocol by selecting the Save this protocol button and then selecting Save in the Saving a Protocol window that appears Select Run with selected plate setup 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 TM270 Page 4 Printed in USA 9 05 Services Help C Edit Protocol Edit Pl

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