PerkinElmer CS Autoplex Analysis Software User Guide
Contents
1. 9 14 2006 2 52 PM Figure 1 1 The PerkinElmer CS Autoplex Analysis Software Main Window The main window includes the following menus and toolbar File a standard Windows menu to open save print and export files and exit the PerkinElmer CS Autoplex Analysis Software Analyze to start the wizard for a selected analysis View to configure the view for the current display a spreadsheet or plot and turn on turn off the display for the toolbar or status bar Help to access online help and version information about the PerkinElmer CS Autoplex Analysis Toolbar buttons provide a shortcut for each task on the File menu Open File Save Save as Save all Print Print Preview and Exit A button for Export to Excel displays when a spreadsheet is open Hold the mouse cursor over a button to display the button name PerkinElmer CS Autoplex Analysis Software i File Analyze View Help PerkinElmer CS Autoplex Analysis Introduction Chapter 1 1 4 Getting Help Help for using the software is available in these ways The PDF file for this user manual is available from the Windows Start menu by selecting Programs PerkinElmer CS Autoplex Analysis Software CS Autoplex Analysis Software User Manual Online help can be accessed from within the PerkinElmer CS Autoplex Analysis Software from the Help menu or by pressing the F1 key to get help for the current window The on line help is also available from t2. Note The map view shown in Figure 2 5 is available only if Plate Location or Both was N selected under the Export Location Label Style on the Luminex IS in the data export options If Sequential was selected the contents will be available only as the list shown in Figure 2 6 Note Tool tips are available for the plate wells in the map view Hold the mouse cursor N over a plate well to see the sample text if it exists or the well information for example Al If you don t see the tool tips you may need to resize the window first ALP TTsTeT7 Te ola A p User Manual 2 9 Chapter 2 Running a Ratiometric Analysis for Gene Ratiometric Analysis Wizard Select Reference Location s Which location s do you want to use as the reference s for the ratio View Contents As OMap Location Sam 1A1 Platei Acquire 2 B1 Platei Acquire 3 C1 Platei Acquire 4 D1 Plate1 Acquire 5 E1 Plate1 Acquire 6 F1 Platei Acquire 7 G1 Plate1 Acquire 8 H1 Platei Acquire 10 B2 Plate1 Acquire 11 C2 Plate1 Acquire 12 D2 Platei Acquire 13 E2 Platei Acquire 14 F2 Platei Acquire 15 G2 Platei Acquire arm minams fan lt Back Next gt Figure 2 6 Select Reference Location s List View 6 After selecting the location s click Next 7 Inthe Summary window review your selections Ratiometric Analysis Wizard Summary Selected Refe
3. 2 3 normalization 2 3 starting 2 7 Reference replicates 2 2 Saved results opening 2 33 Saving spreadsheet results 2 32 Scatter plot display options 2 19 Software installing A 1 B 1 starting 1 1 Spreadsheet table 2 12 exporting to Excel 2 17 grouping results by column 2 12 information provided by 2 13 Starting the software 1 1 Toolbars 1 2
4. Welcome to the PerkinElmer CS Autoplex Analysis Gene Expression Ratiometric Analysis Wizard This wizard helps you configure the ratiometric analysis to be performed on your Luminex xMAP instrument data To view the data without running an analysis click Cancel Click Cancel Click Next to continue Figure 2 2 Cancelling the Analysis Wizard User Manual 2 5 Chapter 2 2 4 1 2 6 Running a Ratiometric Analysis for Gene 5 Click Cancel to view the input data without running an analysis The raw data displays in a spreadsheet table Figure 2 3 AS Note To start an analysis after viewing the data restart the wizard on the Analyze menu select Ratiometric Analysis for Gene Expression to re open the wizard If you have already run an analysis on the current raw data the PerkinElmer CS Autoplex Analysis prompts you to save the results and opens the wizard for an analysis on the existing data Data can be sorted in ascending or descending order for any column by clicking the column header or grouped by columns for easier viewing See The Spreadsheet Table section later in this chapter In addition any columns in the spreadsheet can be hidden or displayed and their order re arranged using the Configure Columns dialog box See Showing or Hiding Columns PerkinElmer CS Autoplex Analysis i File Analyze View Help Ze S I BEN Spreadsheet Scatter Plot Bar Chart Index Location Sample E
5. 5 9 7 10 13 8 5 1 4 8 9 20 10 4 16 10 0 10 11 23 3 5 11 13 5 8 5 3 19 18 5 18 15 11 12 1 6 16 9 24 9 11 15 5 15 13 5 10 5 25 15 5 20 19 19 3 22 5 20 25 25 28 5 25 32 25 29 8271 Save and close the file The file is ready to re open in the CS Autoplex Analysis software Adding Controls Information Using Notepad The batch data may be replayed to include the control keywords IC and EC but it is easier to edit the file manually To add control information 1 2 Open the original Luminex IS file in Notepad All data type data sets need to be modified the easiest way to do this is using replace all On the Notepad Edit menu select Replace In the Replace dialog box specify the string to find the string to replace it and click Replace All Use a unique original string so that the replacement works correctly Save and close the file The file is ready to re open in the CS Autoplex Analysis software PerkinElmer CS Autoplex Analysis Software Glossary Analyte Analyte ID Analyte Name Analyzer Bead bead region bead set control beads Replicates Replicate Samples Luminex xMAP microsphere set microspheres multi analyte qualitative quantitative Sample Sample Name A substance that is detected through assay analysis Each test or bead set tests for a specific analyte
6. A short text name for the analyte or bead region A text description for the analyte or bead region The Luminex 200 100 IS analyzer used to create xMAP files Refers to an xMAP microsphere See microsphere Refers to an xMAP microsphere See microsphere A set of xMAP microspheres that have a uniquely identifiable ratio of two classification dyes The unique ratio is identified by a unique spectral address Used to verify standards Multiple beads with the same analyte in the same concentration having the same ID and name The data set produced using the same sample multiple times in the same bead region Luminex multi analyte microspheres containing a unique mixture of two distinctly colored fluorochromes to distinguish them from other multi analyte microspheres Polystyrene spheres with a diameter in the micrometer range Also called beads Several assays or tests performed simultaneously in the same reaction containers Pertaining to calculations that determine the absence or presence of an analyte Pertaining to calculations that determine the precise numerical measurement of an analyte The mixture of assay components microspheres reporter patient diluent that are analyzed A text description assigned for a sample Standards microspheres assay xMAP Assay standards are substances of known concentrations used to derive a standard curve with which unknown samples and controls are compared to determine
7. S outputs csv W Outputsexi csv My Computer My Documents File name Output3 csv My Network Files of type Luminex IS 2 3 Output csv i 3 In the file list double click the file that you want to open or type the name in the File name field and click Open This opens the data file and automatically opens the analysis wizard Ratiometric Analysis Wizard DAR Welcome to the PerkinElmer CS Autoplex Analysis Gene Expression Ratiometric Analysis Wizard This wizard helps you configure the ratiometric analysis to be performed on your Luminex xMAP instrument data To view the data without running an analysis click Cancel Click Next to continue Figure 2 4 The Gene Expression Ratiometric Analysis Wizard 4 Click Next to continue 2 8 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 5 In the Select Reference Location s window select the location s to use for the ratio To select replicates hold down the Ctrl key as you click each location Ratiometric Analysis Wizard DER Select Reference Location s Which location s do you want to use as the reference s for the ratio View Contents As OList Map 1 2 3 4 5 8 8 9 10 11 12 A A2 7 A3 B1 B2 3 B3 Ci 1 C2 D1 2 D2 E1 3 E2 Fl F2 G1 5 H1 5 lt Back Next gt J Cancel Figure 2 5 Select Reference Location s Map View
8. a new title for the distribution plot and click OK To select a column to display on the distribution plot 1 From the View menu select Distribution Plot Options Select Column Select Column Median io Count IC Mean DO cv C Peak C StdDev E Trimmed Count C Trimmed Mean C Trimmed CV C Trimmed Peak C Trimmed StdDev IC Blank Subtracted Median Gan 2 Select the column to view For distribution plots only one column at a time can be selected The Select All and Clear All buttons are dimmed and unavailable 2 6 6 Setting a Title for a Plot or Chart Before printing or saving a particular view of a chart you may want to change the title to something more meaningful To set a title for a plot or chart view 1 Right click anywhere on the plot or chart The Set Chart Title dialog box opens Set Chart Title Enter the text for the title of this chart De eriments in Well A1 User Manual 2 29 Chapter 2 Running a Ratiometric Analysis for Gene 2 Type a name for the chart and click OK 2 7 Saving the Analysis Results You can save the spreadsheet table as a csv file and save a plot or chart as a BMP GIF PNG JPEG or TIF file Any spreadsheet or plot can be saved individually If you use the Save all command the current view of all open plots can be saved with the spreadsheet Note To save a particular view of a scatter plot bar graph or distribution plot save it ind
9. analytes in a single well The configurable plot lets you select a location to view and any available column to plot The most meaningful data to display would be the data which was used for calculating the resulting ratios The x axis on the bottom displays the selected column data for the reference location If reference replicates were selected the first reference location is displayed on the x axis The y axis on the left displays the selected column data for all analytes in the selected location See Figure 2 10 for an example of a scatter plot The title of the plot displays the name of the currently viewed well location or a title that s been assigned under the Scatter Plot Options described in this section The plot can be printed or saved See Saving the Analysis Results PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 Hold the cursor over a data point to display information PerkinElmer CS Autoplex Analysis Software Eile Analyze View Help SOEBEN readsheet Scatter Plot Bar Chart Distribution Plot Experiments in Well A1 Click to open toolbox uoge307 4D3 aS IC 79CI 4 ALPHA T 12677 500 7393 000 za ueipapy Figure 2 10 Example of a Scatter Plot 2 6 2 1 Selecting a Location to View The PerkinElmer CS Autoplex Analysis provides a toolbox for changing the scatter plot view by location and menu options for configuring the d
10. by editing the data csv file using Microsoft Notepad see Appendix B To edit the spreadsheet for blanks 1 Open the input data file and cancel the analysis wizard 2 Group the rows by location by dragging the Location column header into the group box above the spreadsheet See To group the data by columns on page 2 12 3 Expand each group that corresponds to the blank locations 4 Click any cell in the Sample column This puts the cell in edit mode 5 Modify the text in the cell so that it begins with the word Blank 6 Click in the next row for the Sample column to automatically update all rows for this sample AN Note Information for internal or external controls can be added in a similar same way by editing the spreadsheet and modifying the appropriate Experiment names to begin with IC or EC The data is now ready for analysis From the Analyze menu select Ratiometric Analysis for Gene Expression to open the wizard for analysis 2 6 Reviewing the Analysis Results User Manual The analysis results are displayed in a spreadsheet table in the PerkinElmer CS Autoplex Analysis main window Graphical views of the data including a scatter plot bar chart and distribution plot are also provided in tabbed windows behind the spreadsheet To bring any window to the front click the tab name Chapter 2 Running a Ratiometric Analysis for Gene 2 6 1 The Spreadsheet Table The Spreadsheet shows the an
11. configure the spreadsheet columns 1 On the View menu click Spreadsheet Options Show Hide Columns The Configure Columns dialog box opens The list of columns in this dialog box depends on the data exported to the Luminex Output csv file The following example shows all of the possible columns Configure Columns Check the columns that you would like visible in the results table Use the Move Up and Move Down buttons to reorder the columns Location Experiment wel Median Result Count Mean CV Peak StdDev Trimmed Count Trimmed Mean Trimmed CV Trimmed Peak Trimmed StdDev Ava Result 2 Check the box next to each column that you want to display in the spreadsheet Uncheck the box next to each column that you want to hide Click Show All to check all boxes or check Hide All to uncheck all boxes To re arrange the order of the columns highlight select any column that you want to move and click Move Up or Move Down to move the column to the desired position 2 16 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 2 6 1 4 Flagging Median and Bead Count Values To set the flagging options 1 On the View menu click Spreadsheet Options Flagging Options The Ser Flagging Options dialog box opens The example below shows the default settings Set Flagging Options Flag Median values Minimum Median 100 Maximum Median 20000 Flag Bead Count
12. gt Perkin precisely TM PerkinElmer CS Autoplex Analysis Software User Manual September 2006 Copyright 2006 PerkinElmer Life and Analytical Sciences All rights reserved No part of this manual may be reproduced or transmitted in any form without the written permission of PerkinElmer Printed in the U S A Information in this document is subject to change without notice and does not represent a commitment on the part of PerkinElmer Luminex Luminex 100 Luminex 100 IS and xMAP are trademarks of Luminex Corporation Windows and Microsoft are registered trademarks of Microsoft Corporation Contents Chapter 1 Introduction Overview un nn a Ra hee ee ea 1 1 Starting the SoftWare t e aoai aa ea sn 1 1 The CS Autoplex Analysis Software Window 0 cece ee eee eee eee 1 2 Getting Help u r 4 2 ue cok tote ee Pg ns a a a Pa ates eco Se 1 3 Using the On Line Help u namen aaa Del 1 3 Contacting Technical Support 2 2 ce eee neces 1 3 Chapter 2 Running a Ratiometric Analysis for Gene Expression OVEIVIEW E E EN er nr ERS see en ende 2 1 Input Data for the Analysis 0 0 0c eee etn e eee ned 2 1 How the Analysis Software Handles Data 00 eee ee eee eee 2 2 Reference Replicates si ie sue ISSN Te ain DK 2 2 Blank Subtraction 2 2 08ea ws naar ag Saw as 2 2 Specifying Blank Samples 1 0 0 0 000 cee een teens 2 3 Normalization aa dl aes aan er I nd Med eae hte 2 3 Specifying the In
13. their concentration or quantity See Luminex xMAP microsphere set PerkinElmer CS Autoplex Analysis Index Analysis results bar chart 2 22 distribution plot 2 27 opening saved results 2 33 scatter plot 2 18 spreadsheet table 2 12 Analyze menu Ratiometric Analysis for Gene Expression 2 6 Axes numeric type changing for scatter plots 2 22 changing the Y axis for bar charts 2 25 Axes Ranges changing Scatter and Bar plots 2 23 Bar chart description 2 22 display options 2 23 Blank samples adding information using Notepad B 1 using the analysis spreadsheet 2 11 description 2 2 Blank subtraction ratiometric analysis 2 2 Controls See Normalization B 2 Data for analysis 2 1 2 4 Denominator description 2 2 Distribution plot description 2 27 display options 2 27 Help 1 3 contacting technical support 1 3 User Manual PDF file 1 3 using on line help 1 3 Input data viewing 2 4 viewing the file header 2 6 Installing the software A 1 Main window menus 1 2 menus and toolbars 1 2 Normalization adding controls information using Notepad B 2 using the analysis spreadsheet 2 11 for ratiometric analysis 2 3 Opening input data 2 4 saved results 2 33 PerkinElmer CS Autoplex Analysis software description 1 1 starting 1 1 supported analyses 1 1 Printing spreadsheet or plot 2 32 Ratiometric Analysis for Gene Expression blank subtraction 2 2 described 2 1 2 3
14. Analysis Results Spreadsheet The displayed data columns depend both on the information that was included in the input file and whether any columns are hidden All data that was included in the input file is available for the results If the data was included but a column isn t visible it may be hidden See Display Options for the Spreadsheet on page 2 15 The information can also be grouped by a column to more easily see related data To group the data by columns 1 Click the column heading for example Experiment and drag it into the box above the column headings When the red arrows display above the column headings release the mouse button PerkinElmer CS Autoplex Analysis Software File Analyze View Help HAAG OATS Al preadsheet urn Index Location Sample Experiment well Median Re Al Patient 1 1 TNFRSF1IA Al 15 Al Patient 1 10 BAX Al 207 Al Patient 1 IC 100 CI 1 CYC1 Al 958 2 12 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression 2 2 6 1 1 Chapter 2 The data is grouped by the column and collapsed The example in Figure 2 9 shows the data grouped by Experiment PerkinElmer CS Autoplex Analysis Spreadsheet scatter Piot Bar Chart Experiment 1 TNFRSFLA 27 items Experiment 10 BAX 27 tems P Experiment 11 IGFALS 27 tems Expenment 12 TSSC3 27 items Experiment 13 DARKE 27 Rems Expenment 14 CNTRL NEG 27 Rems Experimen
15. ST Distr Plot bmp II TEST Scatter Plot bmp File name Save as type Windows Bitmap bmp Save Distribution Plot Save in Info My Documents SQ JULY 3 TEST Bar Chart bmp N JULY 3 TEST Dist Plot bmp Sur 3 TEST bmp N July 4dist bmp DS auy 4 bmp I TEST Bar Chart bmp I TEST Distr Plot bmp TEST Scatter Plot bmp File name Save astype Windows Bitmap bmp e a name select the file type and click All of the results are saved and can be re opened for review Saving the Spreadsheet User Manual 2 31 Chapter 2 Running a Ratiometric Analysis for Gene 2 7 2 Saving the Spreadsheet To save the spreadsheet results 1 On the File menu select Save or Save as The Save spreadsheet data dialog box opens Save spreadsheet data Save in Exp Beads Sl copy of Output csv Bah GeneExp csv B output csv E Runoo lcsv Sb Run002 csv My Documents File name Gene bol My Compier Save as type Luminex SW Spreadsheet csv 2 Type a name for the spreadsheet in the File name box and click Save 2 7 3 Saving an a Plot or Chart To save a scatter plot bar chart or distribution plot 1 Display the plot or chart view that you want to save 2 On the File menu select Save or Save as If the view has already been saved it is saved again overwriting the previous file If the view has not been saved the Save as dialog box opens 3 Type a name in the Filename f
16. Viewing the Input Data Opening a valid input data file automatically opens the analysis wizard To view the input data without running an analysis cancel the wizard as described in the following procedure 2 4 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 To open an input data file 1 On the File menu click Open 2 In the Open dialog box navigate to the directory where the input data resides My Recent Documents a My Computer My Network Figure 2 1 Data 3 O8 amp H output7 csv 8 outputs csv FS outputs csv W changed controls Output6 csv FS copy excel2 of Output6 csv Copy excel of Output csv 3S copy from Notepad of Output6 csv 8 output10 csv output11 csv FS copy of Output6 csv W GeneExp 9_11_06 csv SS GeneExp 9_23_06 csv GeneExp Sept 20 csv S outputi csv F output2 csv F output3 csv 8 output4 csv FS Outputs csv W outputs csv E Outputsexi csv OutputS csv v Luminex IS 23 Output csv v File name Files of type Opening a File for Ratiometric Analysis 3 Inthe file list double click the file you want to open or type the name in the File name field In the Files of type field select the type of file Luminex IS 2 3 Output csv 4 Click Open This opens the data file and automatically opens the analysis wizard shown in Figure 2 2 Ratiometric Analysis Wizard
17. alysis Results 2 11 Saving the Analysis Results 2 30 Opening Saved Results 2 33 2 1 Overview The Ratiometric Analysis for Gene Expression calculates the ratio for samples in all well locations One location or a location with replicates is selected as a reference Then the ratio of an analyte in each sample well is compared to the same analyte in the specified reference location s Blank subtraction and normalization can also be used See How the Analysis Software Handles Data for information 2 2 Input Data for the Analysis User Manual Input data for a Gene Expression Ratiometric Analysis must be an Output csv file from a Luminex 100 instrument Configure the assay data for export to the Output csv using the Data Export tab in the Luminex 100 IS Software Options window Refer to the Luminex IS Software Manual for Version V2 3 for information on exporting data The PerkinElmer CS Autoplex Analysis Software supports any data selected for export under the Additional Export Stats on the Data Export tab The following default options are recommended for exporting data Auto Export Batches Plate Location or Both under Export Location Label Style Note that if Sequential is selected the plate map view is not available in the configuration wizard see Figure 2 5 nor in the scatter plot and bar chart results after the analysis Additionally a distribution plot is not created for the results Chapter 2 Running a Ratiometric Anal
18. alysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 User Manual Menu Description Set Title Set Axes Ranges Set Axes Numeric Type Zoom Full Hide Lines Show Lines Opens the Ser Chart Title dialog box Enter a new title for the scatter plot and click OK See Setting a Title for a Plot or Chart on page 2 29 Opens the Set Axes Ranges dialog box Specify the display range of the x and y axes For scatter plots you can set either axis or both axes See Setting the Axes Ranges Scatter Plot or Bar Chart Opens the Set Axes Numeric Type dialog box Here you can specify the numeric type for either axis as Linear is the default or Logarithmic select this if a wide spread in values make it difficult to view all of the data on a linear scale Choose common natural or binary depending on your data and preferences See Changing the Axes Numeric Type Click to return to a full display of the chart after changing the axes ranges This menu selection is dimmed and unavailable if the chart is already at full display Click to hide the lines if they are showing or show the lines if they are hidden The red line represents y x allowing a quick visual determination of which axis has the higher value If a data point is above the red line y gt x If a data point if below the red line y lt x The blue lines are n fold lines that is y n x or y 1 n x In this rele
19. alysis results for all analytes in all locations The customizable view lets you group and sort by column choose which columns to display and flag median and bead count values that fall within a specified range Figure 2 8 is an illustration of a typical results spreadsheet with the default columns showing PerkinElmer CS Autoplex Analysis I File Analyze View Help ISAAA oe 0 Spreadsheet Scatter lot Bar Chart Index Location Sample Experiment Median Result Count Mean Trimmed Count Trimmed Mean E Patienti 1 TNFRSFIA 2 Patient1 10 BAX 295 487 234 841 1 3 Al Patienti IC100 CI 1 CYC1 958 125 997 280 113 972 920 9 4 Al Patienti 11 IGFALS 12 5 164 48 238 148 26 338 1 5 Al Patienti 12 TSSC3 1 142 17 711 128 11 984 6 Al Patienti 13 DARK1 5 155 28 458 141 21 582 7 Al Patienti 14 CNTRL NEG 5 176 27 352 160 19 875 8 Al Patienti 15 LTBR 13 121 48 818 109 28 101 8 9 Al Patienti 16 MCLS 90 123 111 073 111 100 730 7 10 Al Patienti 17 IGFBP4 563 5 126 869 405 114 598 500 5 11 Al Patienti 18 CNTRLNEG2 6 144 32 326 130 25 300 4 12 Al Patient 19 CRADD 93 143 118 469 129 111 419 9 13 Al Patienti 2 BAG1 217 179 270 045 163 242 104 2 14 Al Patienti IC 20 GAPDH 10017 5 110 11023 545 100 10441 040 1 15 Al Patienti 21 BIRC4 5 5 194 40 165 176 29 119 2 16 Al Patienti 22 BINP2 533 154 613 234 140 573 964 5 17 Al Patient 23 BID 17 105 65 571 95 35 916 8 18 Al Patient 24 TNFRSF21 7 5 112 42 063 102 25 020 0 Figure 2 8
20. and Normalized blank data normalized blank subtracted With Normalization median normalized blank subtracted trimmed mean 2 3 1 Reference Replicates Reference replicates are multiple beads incubated with the same sample and assigned the same experiment name or bead ID in the Luminex assay If replicates are selected the PerkinElmer CS Autoplex Analysis software uses the mean of the reference replicates as a denominator for the analysis 2 3 2 Blank Subtraction Blank subtraction uses one or more wells containing sample with zero content to provide a control for non specific signals The measured content of the blank s is subtracted from the measured data in each sample in the following way 2 2 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 If a sample is identified as a blank the software takes the median value of each bead ID in the blank sample well and subtracts it from the median and trimmed mean values of that bead ID in all other wells If replicate blank wells are used the value subtracted is the mean of the medians in the blank samples 2 3 2 1 Specifying Blank Samples To specify one or more blank samples each blank sample must be assigned a name starting with the word Blank when exporting the data to an Output csv file For example a blank sample could be named simply Blank or multiple blank samples could be named as Blank2 Blank3 e
21. art and toolbox side by side N click the pushpin on the toolbox Click the pushpin again to return to Auto Hide 2 6 3 1 Display Options for the Bar Chart To configure the display 1 On the View menu select Bar Chart Options Or right click anywhere on the bar chart to open the options menu User Manual 2 23 Chapter 2 Running a Ratiometric Analysis for Gene 2 From the options menu select the option you want to change The options are described in the following table Menu Description Select Columns Opens the Select Columns dialog box Select one several or all columns of data to display See Selecting Columns for Bar Chart Display on page 2 24 Set Title Opens the Ser Chart Title dialog box Enter a new title for the bar chart and click OK Set Axes Ranges Opens the Set Axes Ranges dialog box Select this option to specify the display range of the x and y axis or to specify a scroll bar for one or both axes See Setting the Axes Ranges Scatter Plot or Bar Chart Set Y Axis Numeric Opens the Y Axis Numeric Type dialog box Here you can specify the Type numeric type for the Y axis as Linear default or Logarithmic if a wide spread in values make it difficult to view all of the data on a linear scale Choose common natural or binary See Changing the Y Axis Numeric Type on page 2 25 Zoom Full Click to return to a full display of the chart This menu selection is dimmed and unavailable if the ch
22. art is already at full display 2 6 3 2 Selecting Columns for Bar Chart Display To select column s to display on the bar chart 1 From the View menu select Bar Chart Options Select Columns Select Columns DER Median Select All Count CV Ratio of Medians to 081 81 Ratio of Means to 081 81 2 Check the box for each column to display Any number of columns can be selected for bar chart display Click Select All to display all columns click Clear All to clear all checkboxes 2 24 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 2 6 3 3 Changing the Y Axis Numeric Type To change the Y axis numeric type 1 With the scatter plot open right click in the plot to open the menu and select Set Y Axis Numeric Type The Y Axis Numeric Type dialog box opens Y Axis Numeric Type x Y Axis Type O Linear Logarithmic Log base Common v Select the axis type Linear or Logarithmic If Logarithmic is selected select a log base from the drop down list Common Natural or Binary Click OK to save the changes 2 6 4 Setting the Axes Ranges Scatter Plot or Bar Chart You can set either or both axes ranges for the scatter plot and bar chart to change the data display or select scroll bars to quickly view data by expanding or collapsing an axis or view data by scrolling along either axis In addition you can choose either linea
23. ase of the analysis software n 2 If a data point is above the top blue line y gt 2 x If a data point is below the bottom blue line y lt 1 2 x 2 6 2 3 Selecting a Column to Display To select a column to display on the scatter plot 1 From the View menu select Scatter Plot Options Select Column Select Column Median Count Mean CV Peak StdDev 00008 Trimmed Count Trimmed Mean Trimmed CV Trimmed Peak Trimmed StdDev Blank Subtracted Median OK Cancel 2 21 Chapter 2 Running a Ratiometric Analysis for Gene Select the column to view For scatter plots only one column at a time can be selected The Select All and Clear All buttons are dimmed and unavailable 2 6 2 4 Changing the Axes Numeric Type To change the axes numeric type 1 With the scatter plot open right click in the plot to open the menu and select Set Axes Numeric Type The Axes Numeric Type dialog box opens Axes Numeric Type X Axis Type Linear O Logarithmic Log base Y Axis Type Linear O Logarithmic Log base 2 For one or both axes select the axis type Linear or Logarithmic 3 If Logarithmic is selected select a log base from the drop down list Common Natural or Binary 4 Click OK to save the changes 2 6 3 The Bar Chart The bar chart displays the values across wells for the user selected analytes or values for all analytes in a single well The configurable chart al
24. d to the Luminex Output csv file Optional The measure of relative dispersion within the distribution CV 100 x Std Dev Mean Optional The value that is equal to the largest number of data points within the distribution For example in data set 1 2 2 3 3 4 5 3 is the peak because it occurs the most number of times in the distribution list Optional The number of data points in the trimmed distribution Nj Optional The sum of the data points in the trimmed distribution divided by the number of data points Trimmed Mean X N Optional The measure of relative dispersion within the trimmed distribution Trimmed CV 100 x Trimmed Std Dev Trimmed Mean Optional The value that is equal to the largest number of data points within the trimmed distribution Optional The measure of dispersion within the trimmed distribution Trimmed Std Dev N X XF X XP Ny Ne 1 Optional The average of any replicate samples final test results based on a qualitative or quantitative analysis PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 User Manual Item Description Analysis Results After analysis the following columns are added to the spreadsheet and are available in the Configure Columns dialog box Note Blank subtracted normalized blank subtracted or normalized data is used for the input column Ratio of lt input column gt
25. distribution web site at www perkinelmer com csautoplex A 2 Installing the Software The software includes an InstallShield Wizard for installing the software To install the software Run the setup exe file to start the software installation User Manual A 1 Appendix A Installing the PerkinElmer CS Autoplex Analysis The InstallShield Wizard installs the software on your computer i PerkinElmer CS Autoplex Analysis Software InstallShield Wizard x Welcome to the InstallShield Wizard for PerkinElmer CS Autoplex Analysis Software The InstallShield R Wizard will install PerkinElmer CS Autoplex Analysis Software on your computer To continue click Next WARNING This program is protected by copyright law and international treaties Figure A 1 The First InstallShield Window Follow the prompts to install the software A 2 PerkinElmer CS Autoplex Analysis Software Adding Blanks and Controls uppendix B Information Appendix Summary Overview B 1 Adding Blanks Information using Notepad B 1 Adding Controls Information Using Notepad B 2 B 1 Overview Information for blank samples and normalization controls should already be included with the data exported from the Luminex software The PerkinElmer CS Autoplex Analysis software uses the sample names beginning with the word Blank to perform blank subtraction and test names beginning with IC or EC for internal or external controls to perfor
26. elect Analyte toolbox open and display the entire plot and toolbox side by side click the pushpin on the toolbox Click the pushpin again to return to Auto Hide To adjust the palette 1 Click Adjust Palette on the left hand side of the Distribution Plot tab The Adjust Palette toolbox opens over the plot Note To keep the Adjust Palette toolbox open and display the entire plot and toolbox side N by side click the pushpin on the toolbox Click the pushpin again to return to Auto Hide 2 Under Low Threshold move the slider to the right to raise the low threshold value or move the slider to the left to lower the low threshold value 3 Under High Threshold move the slider to the left to lower the high threshold value or move the slider to the right to raise the high threshold value Using the two sliders you can effectively narrow the range of values being viewed to those within a certain threshold range 2 28 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 To configure the display 1 Right click anywhere on the distribution plot Or on the View menu select Distribution Plot Options 2 From the options menu select the option you want to change The options are described in the following table Menu Description Select Column Opens the Select Column dialog box Select one column of data to display Set Title Opens the Set Chart Title dialog box Enter
27. figuring an analysis The first supported application is ratiometric calculations for gene expression using Luminex Output csv files The PerkinElmer CS Autoplex Analysis computes the ratios for samples in wells and displays the results in a spreadsheet table scatter plot bar chart and distribution plot 1 2 Starting the Software To start the PerkinElmer CS Autoplex Analysis software Double click the PerkinElmer CS Autoplex Analysis Software icon on the computer screen desktop Wy PerkinElmer CS Autoplex Analysis Software Note If the icon does not appear on the desktop click Start on the Windows task bar then select Programs PerkinElmer CS Autoplex Analysis Software CS Autoplex Analysis Software User Manual The following are also available from the Start menu for the PerkinElmer CS Autoplex Analysis Software CS Autoplex Analysis Software Help CS Autoplex Analysis Software User Manual Uninstall PerkinElmer CS Autoplex Analysis Software Chapter 1 1 3 Introduction The CS Autoplex Analysis Software Window The PerkinElmer CS Autoplex Analysis Software opens to a main window shown in Figure 1 1 The main window is the starting place for each analysis From here you can open and view the input file view file header information start an analysis and view and save analysis results PerkinElmer CS Autoplex Analysis Software i File Analyze View Help SLBA ALS am Spreadsheet
28. he Start menu by selecting Programs PerkinElmer CS Autoplex Analysis Software CS Autoplex Analysis Software Help 1 4 1 Using the On Line Help To use the on line help 1 2 From the Help menu select one of the following Contents to browse the table of contents Click the book icons to display lists of subtopics Index if you know exactly what you re looking for type the word or scroll through the list Search type a word or phrase to search then click List Topics Double click a topic in the search results list to display it To get help for the current task Press the F1 key while in any window of the PerkinElmer CS Autoplex Analysis Software to open the help topic for that window or access any part of the on line help 1 4 2 Contacting Technical Support User Manual To contact technical support Web Site http www perkinelmer com Telephone 800 762 4000 US and Canada or 1 203 925 4602 E mail info perkinelmer com Fax 1 203 944 4904 Worldwide Techsupport perkinelmer com Techsupport europe perkinelmer com 1 3 Chapter 1 Introduction This page is intentionally left blank 1 4 PerkinElmer CS Autoplex Analysis Running a Ratiometric Chapter 2 Analysis for Gene Expression Chapter Summary Overview 2 1 Input Data for the Analysis 2 1 How the Analysis Software Handles Data 2 2 Viewing the Input Data 2 4 Running a Ratiometric Analysis 2 7 Reviewing the An
29. ield and select a file type bmp gif jeg tif or png in the Save as type file 4 Click Save 2 7 4 Printing a Spreadsheet or Plot You can print the current spreadsheet or any of the plots with or without saving them To print a spreadsheet or plot 1 Click the tab for the spreadsheet or for any plot 2 On the File menu select Print or select Print Preview first to see how it looks 3 In the Print window specify the printer and click OK 2 32 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 2 8 Opening Saved Results To open saved analysis results 1 On the File menu click Open The Open dialog box appears 2 Enter the name of the results file in the File name field 3 In the Files of type select the PerkinElmer CS Autoplex Analysis Software Output csv Note The results files will not open unless PerkinElmer CS Autoplex Analysis Software Output is selected as the type of file 4 Click Open The spreadsheet table opens and any plots are regenerated with default settings User Manual 2 33 Chapter 2 2 34 Running a Ratiometric Analysis for Gene This page is intentionally left blank PerkinElmer CS Autoplex Analysis Software Installing the PerkinElmer CS tppenain PA Autoplex Analysis Software Appendix Summary Overview A 1 Installing the Software A 1 A 1 Overview The PerkinElmer CS Autoplex Analysis Software is provided on a
30. iew and provides a palette adjustment tool for screening the results viewed based on high and or low thresholds A Note A distribution plot is available only if the Export Location Label Style selected in the Luminex IS data export options is Plate Location or Both See Input Data for the Analysis on page 2 1 User Manual Hold the cursor over any block to see information about the intensities of the selected analyte The distribution plot as displayed can be printed or saved See Saving the Analysis Results 2 6 5 1 Display Options for the Distribution Plot The distribution plot is configurable so that any column of data can be selected for plotting and any analyte can be selected for viewing The ratiometric data is displayed using a green black red palette as follows Green Indicates a value less than one lt 1 Black Indicates a value of approximately one 1 Red Indicates a value greater than one gt 1 Non ratiometric data is displayed using a green black palette as follows Black indicates low values Bright green indicates high values 2 27 Chapter 2 Running a Ratiometric Analysis for Gene To select a different view 1 Click Select Analyte on the left hand side of the Distribution Plot tab The Select Analyte toolbox opens over the plot 2 Select an analyte from the list The toolbox auto hides when you click anywhere on the plot Note To keep the S
31. isplay for example changing which data column to plot or changing the range of one or both axes User Manual 2 19 Chapter 2 Running a Ratiometric Analysis for Gene To change the scatter plot view 1 Click Select Location on the left hand side of the scatter plot The Select Location toolbox opens over the plot Click the pushpin to keep the toolbox open and display the entire plot at the same time PerkinElmer CS Autoplex Analysis Software i File Analyze View Help Al re AMN Spreadsheet Scatter Plot Bar Chart Pistribution Plot Select Location Experiments in Well A1 View Contents As zg ueIpapy 2 Select a location to view using the list or map The toolbox automatically auto hides when you click anywhere on the plot Note To keep the toolbox open and display the entire plot and toolbox side by side click N the pushpin on the toolbox Click the pushpin again to return to Auto Hide 2 6 2 2 Display Options for the Scatter Plot To configure the scatter plot options 1 On the View menu select Scatter Plot Options Or right click anywhere on the scatter plot to open the options menu 2 From the menu select the option you want to change The options are described in the following table Menu Description Select Column Opens the Select Column dialog box Select one column of data to display See Selecting a Column to Display on page 2 21 2 20 PerkinElmer CS Autoplex An
32. ividually while it is displayed using the Save or Save as command If you select Close before saving the PerkinElmer CS Autoplex Analysis prompts you to save 2 7 1 Using the Save All Command To save all of results 1 On the File menu click Save All The Select Data to Save dialog box opens 2 Check each results format that you want to save and click OK Select Data to Save EIER Spreadsheet cal Scatter Plot iv Bar Chart M Distribution Plot A Save dialog box opens for each tab as follows 3 In the Save Spreadsheet dialog box enter a name and click Save Fie nase zren ne Suve atiype Lumine SW Spenasshnet f esv 2 30 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 4 Inthe Save Scatter Plot dialog box enter a name select the file type and click Save Save Scatter Plot Save in rfo My Recent Documents Desktop My Documents My Computer My Network 5 Nj ULY 3 TEST Bar Chart bmp N JULY 3 TEST Dist Plot bmp ur 3 TEST bmp I July 4 dist bmp July 4 bmp NV TEST Bar Chart bmp I TEST Distr Plot bmp TEST Scatter Plot bmp File name Seveasype MEERE Save in Info My Documents 6 Inthe Save Distribution Plot dialog box typ Save SQ JULY 3 TEST Bar Chart bmp DULY 3 TEST Dist Plot bmp NJ JULY 3 TEST bmp N July 4 dist bmp N July 4 bmp I TEST Bar Chart bmp I TE
33. lows selecting which columns of data to display and which analyte or location to view Select one several or all columns for the display The legend on the right displays the color that represents each column The title of the chart displays the name of the selected analyte or well location or a title that s been assigned under the Bar Chart Options described in this section The bar chart can be printed or saved See Saving the Analysis Results 2 22 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 Figure 2 11 is an example of a bar chart Hold the cursor over any data point to display information PerkinElmer CS Autoplex Analysis Experiments in Well 3 C1 127635 Selection s 349 View Contents As Ouist Map A A A A A B B B gt B occ 2 D D D E E E F F F dir 6 G G 1TNFRSFIA 10BAX IC100CI1CYC1 11 IGFALS 121853 H H H Figure 2 11 Example of a Bar Chart To change the bar chart view 1 Click Select a View on the left hand side of the bar chart The Select a View toolbox opens over the chart 2 Select Analyte as the view and select an analyte from the list or Select Location as the view and select a location from the list or map The toolbox automatically auto hides when you click anywhere on the bar chart Note To keep the toolbox open and display the entire ch
34. m normalization If the information is not in the data file it can be added by editing the data file in one of two ways The data file can be modified using Microsoft Notepad as described in this appendix The data file can be modified using the spreadsheet within the PerkinElmer CS Autoplex Analysis Software See Adding Blanks or Controls Information after Acquisition on page 2 11 AN Note Do not edit the data file in Microsoft Excel Excel modifies the data formatting when saving the file making the file unusable by the PerkinElmer CS Autoplex Analysis software B 2 Adding Blanks Information using Notepad User Manual The batch data may be replayed to include the keyword that indicates a blank sample However it is easier to edit the file by modifying the sample name strings for each blank Only the Median data rows need to be modified To add blanks information 1 Open the original Luminex IS file in Notepad Appendix B B 3 Adding Blanks and Controls Information Find each blank well location and modify the Sample name to begin with the word Blank In the following example the well location C4 was located in the file and the sample name changed to blank C4 Blank 2 5 5 10 34 5 0 4 11 5 3 5 7 11 0 13 1 0 8 5 0 8 17 7 4 0 35 7 17 3 0 7 5 12 3736 9 117 8 9 4 57 35 3 0 15 3 7 11 14 5 10 11 5 8 2 5 13 5 6 3 24
35. n place of raw values in each of the four steps 2 3 3 1 Specifying the Internal or External Controls Control beads are indicated by assigning a name beginning with IC or EC to the experiment name bead ID when creating the Output csv export file after the Luminex assay Refer to the Luminex 100 IS Developer Workbench Guide V2 3 for information on adding test names 2 3 Chapter 2 Running a Ratiometric Analysis for Gene 2 3 3 2 Enabling Normalization Normalization is off by default and must be enabled by checking the box in the Normalization Options dialog box Once selected the Normalization Options remain in effect until changed To specify normalization for an analysis 1 On the View menu select Normalization Options Normalization Options Perform Normalization Normalization to Controls Gan 2 In the Normalization Options dialog box check the box for Perform Normalization to have the data normalized or uncheck the box to skip normalization 3 Check the box for Normalization to Controls if internal or external controls have been included in the assay specified in the data by a name beginning with IC or EC If Perform Normalization is checked but Normalization to Controls is not checked the analysis uses Normalize to Total If both Perform Normalization and Normalization to Controls are checked but no controls are defined in the data the analysis uses Normalize to Total 2 4
36. ntly open file Header of lt unsaved results gt HER Maximize button Field Name Field Value Program Luminex 100 IS Build 2 3 Date 8 30 2006 11 45 SN LX10005312402 Session Batch 8 22 2006 1 Operator MDeviin DDGate 7000 to 18000 Samples 27 2 To see the full pathname for the data file click the maximize button or resize the dialog box by dragging the bottom right corner 2 5 Running a Ratiometric Analysis Use the Gene Expression Ratiometric Analysis wizard to configure the settings for the analysis The wizard opens automatically when the correct type of input file is opened Note If you already opened the input file and cancelled the wizard to view the data use the Analyze menu to start the wizard Select Analyze Ratiometric Analysis for Gene Expression To run a Gene Expression Ratiometric Analysis 1 On the File menu click Open User Manual 2 7 Chapter 2 Running a Ratiometric Analysis for Gene 2 In the Open dialog box navigate to the directory where the input data resides in Data Be W changed controls Output6 csv H output7 csv FS copy excel2 of Output6 csv W outputs csv My Recent BS copy excel of Output6 csv 8 outputs csv Documents copy from Notepad of Output6 csv Output 10 csv FS copy of Output6 csv S output11 csv W GeneExp 9_11_06 csv 8 GeneExp 9_23_06 csv W GeneExp Sept 20 csv S outputt csv SB output2 csv F output3 csv W output4 csv FS output5 csv rg
37. r or logarithmic as the numeric type for the scatter plot axes or the bar chart y axis To set the axes ranges 1 User Manual Click the tab for the scatter plot or bar chart Chapter 2 Running a Ratiometric Analysis for Gene 2 Right click in the plot to open the menu and select Set Axes Ranges The Set Axes Ranges dialog box opens Set Axes Ranges EIER X Range Automatic O Manual Ouse Scroll Bar Y Range Automatic O Manual Ouse Scroll Bar 3 Refer to the following table and set the ranges Click OK Item Description Y Range or X Range Each axis range has the following range options Automatic Select to have the PerkinElmer CS Autoplex Analysis software automatically set the range for the axis Manual Select to specify the range manually enter the values Minimum Enter a value for the range minimum it must be lower than the maximum range value Maximum Enter a value for the range maximum Use Scroll Bars Select to add scroll bars that can expand or compress and lets you scroll through the axis to better view the data Available for bar charts and scatter plots 2 26 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 2 6 5 Distribution Plot The Distribution Plot shows the distribution of values for the selected analyte across all wells The configurable plot allows selecting which column of data to display which analyte to v
38. rence Locations The following locations are used for blank subtraction Sample Blank in location C2 Sample Blank in location C4 The following controls are used for normalization Internal Controls IC 100 CI 1 CYC1 IC 20 GAPDH IC 6 CI 2 NUP62 IC 71 CI 3 RPS9 IC 79 CI 4 ALPHA T To close the wizard click Finish Figure 2 7 Summary Window To change any of the selected location s click Back once to return to the Select Reference Location s window 2 10 PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 e To change normalization selections you must cancel the analysis set the Normalization Options from the View menu then run the analysis from the Analyze menu AN Note If existing blank wells were not identified during the acquisition the information is not available in the input file for the analysis The blanks information can be added to the input data from within the PerkinElmer CS Autoplex Analysis software using the spreadsheet table Cancel the analysis and edit the data See Adding Blanks or Controls Information after Acquisition 8 If you re satisfied with the selections click Finish to start the analysis 2 5 1 Adding Blanks or Controls Information after Acquisition There are two ways to add the blanks information to the raw data file by editing the spreadsheet from within the PerkinElmer CS Autoplex Analysis software see below or
39. t 15 LTBR 27 tems u Experiment 16 MCLS 27 items Expenment 17 IGFBP4 27 tems Experiment 18 CNTRL NEG 2 27 items E Experiment 19 CRADO 27 Items Expenment 2 BAGI 27 items Experiment 21 BIRCH 27 Rems Expenment 22 BINPZ 27 tems Experiment 23 BID 27 items u Experiment 24 TNFRSF21 27 Items s Expenment 25 8CL10 27 Rems Experiment 26 TNFSE21 27 items Experiment 27 TP53 27 tems Expenment 28 TRAFI 27 items E Experiment 29 AKTI 27 Items Expenment 3 DFFB 27 items lt Figure 2 9 Data Columns Grouped by Experiment To see all information for a grouped column click the plus sign to expand the selection Click again to collapse it For example an Experiment when expanded shows all locations for that experiment To return to the full display of the columns drag the column heading back down to the spreadsheet headings row Information Provided in the Spreadsheet Table The following information is included in the spreadsheet for analysis results Median is always included as well as the calculated Ratio of Median or its equivalent after the analysis Ratio of Trimmed Mean or its equivalent is included in the spreadsheet after analysis only if the trimmed mean data was included in the input file If replicates are selected the denominator is the mean of the replicates Outliers are not removed Additional columns are included depending on
40. tc You may enter this information manually in the Luminex IS software or by using a patient list in the Luminex IS software Refer to the Luminex IS Software Manual for Version V2 3 for details for entering Sample ID information 2 3 3 Normalization User Manual Normalization if enabled corrects the intensity of each signal with respect to the control s The PerkinElmer CS Autoplex Analysis software uses Normalize to Total to all beads unless one or more internal and or external controls have been specified in which case the analysis software normalizes to the specified control s If both internal and external controls have been defined both are used for calculating the normalization factors The Ratiometic Analysis software calculates the normalization using the following method 1 The software calculates the mean of the median values from all wells for each bead region identified as a control which could be all beads 2 Within each well the software calculates the ratio of the median value for each bead to it s corresponding mean value determined in step one 3 Within each well the software calculates the mean of the ratios determined in step two to determine the normalization factor for the well 4 The normalized value is the median or trimmed mean value for each bead in a well divided by the well normalization factor determined in step three If blank subtraction was performed blank subtracted values are used i
41. ternal or External Controls 0 0 0c cece ees 2 3 Enabling Normalization 000 00 cet ee nee nee 2 4 Viewing the Input Data 0 eect ene n een e nee 2 4 Viewing the File Header Information 0 0 0 c cece eee eee 2 6 Running a Ratiometric Analysis 0 0 cece cect ene n eens 2 7 Adding Blanks or Controls Information after Acquisition 2 11 Reviewing the Analysis Results 22 0 0 0 cece cece ete ee eens 2 11 The Spreadsheet Table ag eset sete sot othe date oad hontai 2 12 Information Provided in the Spreadsheet Table 22222 2 13 Display Options for the Spreadsheet 0 0 0 0 06 ccc cece 2 15 Showing or Hiding Columns 600 teen nes 2 16 Flagging Median and Bead Count Values 0 000 cece 2 17 Exporting the Spreadsheet to Excel 0 0 6 ccc ccc eens 2 17 The Scatter Plot gt 25 2282 Geld ape ee BE Soa ds EB es 2 18 Selecting a Location to View 6 ence eens 2 19 Display Options for the Scatter Plot 0 00 ccc cen 2 20 Selecting a Column to Display 0c c ccc eens 2 21 Changing the Axes Numeric Type 1 00 ccc eens 2 22 The Bar Chart ass nm esse ne ml 2 22 Display Options for the Bar Chart 2 2222 cence 2 23 Selecting Columns for Bar Chart Display 22222222 eeeeeene nn 2 24 Changing the Y Axis Numeric Type 0 000 ene 2 25 Setting the Axes Ranges Scatter Plot or Bar Chart 000 2 25 Distribu
42. the input data Refer to Table 2 1 for a description of the possible data columns If some columns aren t visible in your spreadsheet they may be turned off in the Configure Columns window or the data may not have been included in the input file User Manual 2 13 Running a Ratiometric Analysis for Gene Table 2 1 Spreadsheet Table Columns Item Description Input Data Location Sample Experiment Count Median The following columns for input data are available Location of the sample in terms of the command list sequence 1 2 3 etc plate location A1 B1 C1 etc both 1 A1 2 B1 3 C1 etc Or advanced batch 1 A1 Plate1 Acquire 2 B1 Plate1 Acquire etc The name of the sample as defined in the batch setup in the Luminex 100 IS analyzer The name is limited to 30 characters This is the test name from the Output csv file This name defaults to the bead ID but it can be any name entered when setting up the assay on the Luminex instrument The number of data points in the distribution n The number of gated events that fell within the test s specified region The middle value in the distribution of data Additional Export Stats CV Peak Trimmed Count Trimmed Mean Trimmed CV Trimmed Peak Trimmed Std Dev Avg Results Columns for any of the following information are included after analysis if the data was included with the input data exporte
43. tion Plotas 2 2 8 ee ee ae ls ar dl 2 27 Display Options for the Distribution Plot 22222220 cee 2 27 Setting a Title for a Plot or Chart 2 0 eee eee eee 2 29 Saving the Analysis Results 2 2 0 0 0 eee eect cent ence 2 30 Using the Save All Command 00 cece eee eee eee 2 30 Saving the Spreadsheet 0 eee eee eee nensi 2 32 Saving an a Plot or Chart 0 2 eee cnc eee ee 2 32 Printing a Spreadsheet or Plot 0 0 2 eee eee eee eee 2 32 Opening Saved Results 4 2 22 ee ae 2 33 Appendix A Installing the PerkinElmer CS Autoplex Analysis Software OVEIVIEW nennt a aA a da des a Sad weeds sun ein A 1 Installing the Software ssrt ers 4074 p40 u ee er onen Ge Paes A 1 Appendix B Adding Blanks and Controls Information OVENI EM ee a e u ER A E E a N a B 1 Adding Blanks Information using Notepad 00 0 cece eee eens B 1 Adding Controls Information Using Notepad 0 0 0 cece eee ee eee ee B 2 Glossary Index Introduction Chapter 1 Chapter Summary Overview 1 1 Starting the Software 1 1 The CS Autoplex Analysis Software Window 1 2 Getting Help 1 3 1 1 Overview The PerkinElmer CS Autoplex Analysis software analyzes Luminex xMAP output data for various high volume life science and diagnostic applications For each type of application the PerkinElmer CS Autoplex Analysis provides a separate software plug in each with an easy to use wizard for con
44. to lt well Or where e the lt input columns is one of the following Median or Trimmed Mean and the lt well gt is the first reference well that was selected for the ratio in the wizard For example Ratio of Median to C1 Ratio of lt input column gt to lt well gt and replicates where e Ifthere are replicates the column title includes and replicates the replicates are not specified by name For example Ratio of Median to C1 and replicates 2 6 1 2 Display Options for the Spreadsheet Display options for the spreadsheet include hiding columns and flagging data that fits within a specified range To configure the spreadsheet options On the View menu select Spreadsheet Options right click anywhere on the spreadsheet to open the options menu From the menu select the option you want to change The options are described in 1 or 2 the following table Menu Description Show Hide Columns Show Header Flagging Options Select data columns to hide or display and arrange them in order See Showing or Hiding Columns on page 2 16 View information for the current data file See Viewing the File Header Information on page 2 6 Specify to flag median values and bead count values that fall above or below specified values See Flagging Median and Bead Count Values on page 2 17 2 15 Chapter 2 Running a Ratiometric Analysis for Gene 2 6 1 3 Showing or Hiding Columns To
45. values Minimum Bead Count 50 2 Set the options and click OK The following table describes the options Item Flag Median Values Flag Bead Count Values Description Check this box to flag the median values with a background color in the spreadsheet Minimum Median Value leave the default or specify a new value Pick Color click to open the Color dialog box and select a different color Click a color and click OK Maximum Median Value leave the default or specify a new value Pick Color click to select a different color for flagging Check this box to flag the bead count values that are above the specified value Minimum Bead Count leave the default or specify a new value Pick Color click to select a different color for flagging 2 6 1 5 Exporting the Spreadsheet to Excel To export the spreadsheet table to Excel 1 On the File menu select Export to Excel or click the Toolbar icon 2 In the Save as dialog box enter a name in the File name field User Manual 2 17 Chapter 2 Running a Ratiometric Analysis for Gene 3 In the Save as type field select MS Excel Worksheet xls and click Save Save As Save in Exp Beads 5 bead BS LuminexData2 xis Bl LuminexData xis it Documents File name LuminexData062204 My Computer Save as type MS Excel Worksheets 2ds 2 6 2 The Scatter Plot Data in a scatter plot shows the values for all
46. xperiment Median Result Count Mean Trimmed Count Trimmed Mean E Patienti 1 TNFRSFIA 2 Patienti 10 BAX 295 487 107 234 841 1 3 Al Patienti IC100 CI 1 CYC1 958 125 997 280 113 972 920 9 4 Al Patienti 11 IGFALS 12 5 164 48 238 148 26 338 1 5 Al Patienti 12 TSSC3 1 142 17 711 128 11 984 6 Al Patienti 13 DARK1 5 155 28 458 141 21 582 4 7 Al Patienti 14 CNTRLNEG 5 176 27 352 160 19 875 8 Al Patienti 15 LTBR 13 121 48 818 109 28 101 8 9 Al Patienti 16 MCLS 90 123 111 073 111 100 730 7 10 Al Patienti 17 IGFBP4 563 5 126 869 405 114 598 500 5 11 Al Patienti 18 CNTRLNEG2 6 144 32 326 130 25 300 12 Al Patienti 19 CRADD 93 143 118 469 129 111 419 9 13 Al Patient 2 BAG1 217 179 270 045 163 242 104 2 14 Al Patienti IC 20 GAPDH 10017 5 110 11023 545 100 10441 040 1 15 Al Patienti 21 BIRC4 5 5 194 40 165 176 29 119 2 16 Al Patienti 22 BINP2 533 154 613 234 140 573 964 5 Figure 2 3 Input Data for an Analysis Viewing the File Header Information The file header includes information such as the program used and the date that the input data was created and by whom This information does not display in the spreadsheet table but is always available for viewing PerkinElmer CS Autoplex Analysis Software Running a Ratiometric Analysis for Gene Expression Chapter 2 To view the file header information 1 On the View menu click Spreadsheet Options Show Header The Header of dialog box opens to display header information for the curre
47. ysis for Gene 2 3 How the Analysis Software Handles Data This section describes how the PerkinElmer CS Autoplex Analysis software handles input data for the analysis Reference replicates blank subtraction and normalization are supported These are described below If reference replicates are used the mean of the reference replicates is used by the software as the denominator for analysis Outliers are not removed Prior to calculating the ratio during analysis the CS Autoplex Analysis software checks the numerator sample value and denominator the reference value and uses one of the following If either numerator or denominator value is less than one lt 1 the analysis uses the value of 1 as follows If the numerator is less than one lt 1 the ratio will be denominator If the denominator is less than one lt 1 the ratio is the numerator 1 If both the numerator and denominator are less than one lt 1 the ratio will be 7 7 During analysis the software uses the following data Data Options Data used for Ratiometic Calculations No blank subtraction and Raw data median trimmed mean No normalization With Blank subtraction and Blank subtracted data blank subtracted median blank No Normalization subtracted trimmed mean No Blank subtraction and Normalized data normalized median normalized trimmed With Normalization mean With Blank subtraction
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