Software. Version Code TP700/TP760. Quick Reference TAKARA BIO INC.

Transcription

1 TM Software Version 5.00 Code TP700/TP760 Quick Reference TAKARA BIO INC.

2 NOTICE P10 PCR Notice Purchase of this instrument conveys a limited non-transferable immunity from suit for the purchaser s own internal research and development and applied fields other than human in vitro diagnostics under one or more of U.S. Patents No.5,475,610 or corresponding claims in their non-u.s. counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or by estoppel under any other patent claim. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Applied Biosystems does not guarantee the performance of this instrument L45 Heated Cover This product is covered by the claims of U.S. Patent 5,552,580, and their corresponding foreign counterpart patent claims. L47 Real-Time PCR Quantification Method The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627, 6,303,305, and 6,503,720, owned by the University of Utah Research Foundation and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH.

3 TM Table of contents Experiment workflow 2 1.Startup and shutdown 3 2. Initial screen and experiment type 4 3. Main screens of experiment file 6 4. Thermal Profile Setup (1)Program the PCR conditions 9 (2)Start run and run status 12 5.Plate Setup (1)Basic operations 13 (2)Settings of Relative Quantification 16 (3)Settings of other experiment types 19 6.Result/Analysis (1)Basic operations 22 (2)Graphs and analysis 23 7.Export the results (1)Export graphs and data 30 (2)Export files for RDML 32 8.Appendix 33 1

4 Experiment Workflow Startup the Thermal Cycler Dice TM Real Time System Lite 1) Startup the instrument Turn ON the power on/off switch at the bottom right of the front panel. 2) Startup the computer 3) Launch the software Double-click the shortcut icon on the desktop to launch the software. Experiment File setting 4) Create a new Experiment File Select [File] > [New] and set the Experiment Type and User ID. 5) Plate Document setting Enter the sample information in the Plate Setup screen. 6) Define PCR conditions Define PCR conditions in the Thermal Profile Setup screen. Prepare the reaction plate and Start run 7) Prepare the reaction plate/tubes 8) Set the plate/tubes into the instrument Lift up the lid handle and set the reaction plate/tubes on the sample block. 9) Start run Click Start Run button in the Thermal Profile Setup screen. 10) Monitor the fluorescence data Fluorescence data is displayed in the Result/Analysis screen. Data Analysis 11) Analysis setting Adjust the baseline and threshold. 12) Export results Shutdown the system 13) Terminate the software 14) Shutdown the computer 15) Shutdown the instrument Turn OFF the power on/off switch at the bottom right of the front panel. 2

5 1.Startup and Shutdown Startup a. Turn ON the power on/off switch. Turning the power on leads to warm up the lid heater about 5 minutes until the temperature is higher than 100 while the STANDBY indicator is blinking. After the warming up is completed, the indicator lights steadily and the machine is ready to use. STNDBY indicator b. Start up the computer. c. Double-click the shortcut icon on the desktop to startup the software. Power on/off switch d. Confirm that the instrument and camera status are displayed as "Connected" in the status bar. [Note] If the instrument is not connected with the computer, the status is displayed as Disconnected". Shutdown a. Terminate the software. b. Shut down the computer. c. Shut down the instrument. 3

6 2.Initial screen and experiment type Initial screen The software starts with the initial screen like a right figure. Create a new experiment file to do a new experiment. Or open an existing file to analyze it. Create a new experiment file Click the New experiment icon or select [File] > [New] from the menu bar. Open an existing experiment file Click the Open experiment data icon or select [File] > [Open] from the menu bar. Open experiment data New experiment Experiment type setting New Experiment Options window is displayed when you create a new experiment file. Select the Experiment Type and User ID. Absolute Quantification: In the Absolute Quantification, the copy number of the template is determined using a standard curve. There are four kinds of sample type that can be set; they are Unknown (UNKN), Standard (STD), No Template Control (NTC), and No Amplification Control (NAC). Relative Quantification: In the Relative Quantification, the change of gene expression level of a test sample relative to a control sample is determined. There are two calculation methods used for relative quantification; standard curve method and ΔΔCt method. In this experiment type, Reference (REF; housekeeping gene used to normalize the amount of RNA) and Calibrator (control sample) can be set in addition to the items of the Absolute Quantification. Plus/Minus Assay: Plus/minus judgments are done for the Target and the Internal Control respectively. Those results are combined to make an integrated judgment of Plus/Minus for the unknown sample. In this experiment type, Unknown (UNKN), Positive Control (PC), and Negative Control (NC) can be set. 4

7 SNP Genotyping Assay: Plus/minus judgments are done for the Allele1 type and the Allele2 type respectively. Those results are combined to make an integrated judgment of Genotype for the unknown sample. In this experiment type, Unknown (UNKN), Positive Control (PC) Allele1, Positive Control (PC) Allele2, and Negative Control (NC) can be set. Multiplex-PCR: In a multiplex-pcr experiment, more than two kinds of genes are amplified and detected in one tube using different kinds of fluorescence. Single-PCR: In a single-pcr experiment, only one gene is detected in one tube using one kind of fluorescence. Change the experiment type You can change the Experiment Type after the run is completed. In this case, some sample information may be lost, so you might have to do the plate setup again. a. Select Experiment Options screen. b. Click Edit button in the Experiment Type field to change the settings. c. Click Update button to apply the changes of settings. * Data Polarity setting This setting can be used for the analysis of a detection method in which fluorescent intensity decreases according to the increase of PCR products. In this case, you should select "-". The plus/minus of amplification plots will be inverted and you can analyze the data as usual. 5

8 3.Main screens of experiment file There are five main screens, and you can switch the screens with the buttons on the left side. Experiment Options Set the Experiment Type and User ID in this screen. Description is a free space you can note experimental information etc. (See the previous page for the screen image.) Plate Setup Set the sample information in this screen. First, make a Target List and Sample List, and then set Target and Sample for each well on the Plate Image. 6

9 Thermal Profile Setup Program the PCR conditions and select which filter to use. After that, start the run. Result/Analysis In this screen, data for the current run can be monitored in real time. After completion of the run, the data is analyzed and the results are displayed. Export results, if desired. 7

10 Output Setup Select files you need for making of RDML file, and export them. * This screen is only for Absolute Quantification and Relative Quantification. 8

11 4.Thermal Profile Setup Select the fluorescent filter and program the PCR conditions in the Thermal Profile Setup screen. After that, you can start the run and check the run status on this screen. [Note] You don't have to fully setup the Plate Document (refer to page) before you start the run, because you can set or change it during and after the run. Make sure to set the correct fluorescent filter and PCR conditions when you start the run. (1) Program the PCR conditions Select the type of filter to detect Select the type of filter to detect in the Data Collect box at the top left of the screen. In default setting, both FAM and ROX are selected. If one of them is not needed, remove the check of the filter. In SYBR Green assay, it is recommended to select only FAM without selecting ROX. * FAM; Carboxyfluorescein fluorescent dye, ROX; Carboxy-X-rhodamine fluorescent dye [Note] The instrument will not collect and save the data of unchecked filter(s). It is not possible to change this setting and re-analyze the data after the run. Before you start, make sure to select the correct fluorescent filter(s). It is not recommended to select both of the filters all the time, because detecting with two filters takes longer than one filter. Program the PCR conditions The default PCR conditions are below. Change them, if necessary, to the appropriate conditions in your experiment. AQ, RQ PM, SNP Hold (pre-heat) Hold (pre-heat) 95 C, 30 sec. 95 C, 10 or 30 sec. 2 Step PCR: 40 cycles 3 Step PCR: 45 cycles 95 C, 5 sec. 95 C, 5 sec. 60 C, 30 sec.* (Data Collect) 55 C, 10 sec. 72 C, 20 sec.* (Data Collect) *Primer design or sequences to be amplified may affect amplification efficiency. The amplification efficiency may be improved by increasing the elongation time up to 1 min. Please refer to user manuals or protocols of the PCR reagents to use. [Change the cycle number, temperature and time] Double-click in each column cell that you want to change and enter appropriate numbers or click on /. 9

12 [Add Pattern] At first, select the segment where you want to insert a new pattern. Next, select a pattern from the list at the bottom of this screen and click the Add Pattern button. For example, when you want to add Dissociation Curve after 2 Step PCR... At first, select the Segment 2 of 2 Step PCR. Select with this bar Next, select Dissociation from the list and click Add Pattern button. Now, Dissociation protocol is added after 2 Step PCR. 10

13 [Add Segment] You can add segments in the same way as patterns. Select the place where you want to add a segment and click the Add Segment button. Custom is the only pattern you can add segments to. You can't add segments to Hold, 2 Step PCR and 3 Step PCR. [Delete Pattern or Segment] Select the pattern(s) or segment(s) you want to delete and click the Delete button. You can delete segment(s) of Custom and all kinds of pattern(s). You can't delete segment(s) of Hold, 2 Step PCR and 3 Step PCR. Load template from other file If you will perform a run with the same condition as you did before, you can import the Thermal Profile Setting from other existing experiment file. Click the Load Template button on the top right of the screen and select the file you want to import. [Note] When you load the template, the setting of "Data Collect" and "Dissociation Time" are also imported. Extended menu If desired, you can use the extended menu. Select the Extend at top-right of the screen. Change the Ramp Rate The instrument usually operates at the maximum heating and cooling speed. You can change the ramp rate from 0.01 to 0.07 C per second, if desired. Time Extension If the Hold Time is to be extended for each cycle, input the extension time into the Increment Time field. Temperature Change If the temperature is to be changed for each cycle, input the temperature increment / decrement in degrees in the Increment Temp field. 11

14 (2) Start run and run status Start run After setting the PCR conditions, set the reaction plate/tubes onto the sample block of the instrument. Click the Start Run button in the lower right of the screen to start the run. In case the experiment file is not saved, the run will be started after saving it. The lid handle of the instrument will be locked and you can't open it during the run. Run status During the run, you can check the status on the left side of the screen. In default, Time/Temperature tab is selected and the remaining time and temperatures are displayed. If you want to check the Pattern, Segment and Cycle executing now, click the Running Point tab. Instrument Control In the Instrument Control tab, there are buttons to control the instrument during the run. Add Cycle (Add cycle number during run) Pause (Stop run temporarily) Restart (Restart run that was paused) Stop (Stop run) Skip to Next (Stop the pattern now executing and skip to next pattern) 12

15 5.Plate Setup Enter the sample information in the Plate Setup screen. The information set here is reflected to the graphs in the Result/Analysis screen. You can do the Plate Setup either before the run, during the run or after the run. It is also possible to change the setting and perform the analysis again. (1) Basic Operations a. Making of Target List and Sample List Enter or select information in the Target List and Sample List. The asterisk (*) means those are essential items (Type*, Name*, etc.). Click Add button to increase Targets or Samples, and click Delete button to decrease them. [Note] Load List You can load Target List and Sample List that you made outside this software. Those lists should be TSV (Tab Separated Values) text file. Click the Load button and select the file you want to import. [Note] Set the Target/Sample Type at one time If multiple targets or samples have the same Type, you can set it at one time. First, select all targets or samples with the same Type, and then select the Type from drop-down menu. b. After completing the lists, click Update button. 13

16 c. Making of Plate Image Set the Target and Sample for each well on the Plate Image. (1) Select the corresponding wells. (2) Select the Target or Sample for the wells on the Plate Image Editor. Change ID/Name display (1) Select wells (2) Select Target or Sample After you finish the setting, click Hide Editor button to close the Plate Image Editor. At that time, Hide Editor button will change to Show Editor button. Click the button again to re-open the Plate Image Editor. [Note] Copy, Paste and Delete sample information You can copy and paste the sample information. And you can also undo or redo the operation. To delete the sample information, select the corresponding wells and press the delete key on the keyboard. Copy: Ctrl + C Undo: Ctrl + Z Paste: Ctrl + V Redo: Ctrl + Y [Note] ID/Name Display Although sample information is displayed with names in default, you can also choose the display with IDs instead of names. 14

17 Auto Target/Sample You can use Auto Target/Sample function to do the setting continuously. a. Set the first Target or Sample as usual. b. Click the Auto Target or Auto Sample button to activate the Auto function. c. During the Auto function is ON, the next ID is displayed at mouse pointer. d. Select the corresponding wells to set the next Target/Sample automatically. e. To stop the Auto function, click the Auto Target or Auto Sample button again. c. The next ID at mouse pointer. This ID will be set when you select wells. Load template If you will perform a run with the same plate setup as you did before, you can import the setting from other existing experiment file. Click the Load Template button on the right top of the screen and select the file you want to import. [Note] You can't load the plate setup from other kind of experiment type files, for example from a relative quantification file to an absolute quantification file, because the items that can be set are different. 15

18 (2) Settings of Relative Quantification There are two kinds of quantification methods by real-time PCR, absolute quantification and relative quantification. In addition, the relative quantification includes the standard curve method and the ΔΔCt method. Absolute Quantification Relative Quantification (Standard curve method, ΔΔCt method) In this section, settings of relative quantification (standard curve method) are described. For other experiment types, refer to (3) Settings of other experiment types. The following figure is an example of plate format for gene expression analysis. There are three genes to be analyzed and one of them "Gapdh" is a reference gene to normalize the amount of RNA in each samples. And there are four samples, one is control (In this software, it is called Calibrator) and three of the rest are unknown samples. The quantity of the unknown relative to calibrator is calculated as a result A NTC STD1 STD2 STD3 STD4 STD5 STD6 STD7 B Liver Stomach Kidnay Brain C NTC STD1 STD2 STD3 STD4 STD5 STD6 STD7 D Liver Stomach Kidnay Brain E NTC STD1 STD2 STD3 STD4 STD5 STD6 STD7 F Liver Stomach Kidnay Brain Gapdh Pah Sdh1 Target (Gene) Sample Gapdh: REF (Reference) Liver: Unknown (Calibrator) Pah: TOI (Target of Interest) Stomach: Unknown Sdh1: TOI (Target of Interest) Kidney: Unknown Brain: Unknown a. Making of Target List (1) Select the Type from the pull down menu. REF (Reference): Reference gene to normalize the amount of RNA (housekeeping gene) TOI (Target Of Interest): Target gene to be quantified (2) If needed, change the Dye. Select from the pull down menu. (3) Enter the Target Name. (4) If needed, change the Color. 16

19 b. Making of Sample List (1) Select the Type from the pull down menu. NTC (No Template Control): Control reaction performed without template. NAC (No Amplification Control): Control reaction performed without enzyme (RTase or DNA polymerase). STD (Standard): Sample of known concentration to make a standard curve. UNKN (Unknown): Sample of interest whose quantity will be determined. (2) Enter the sample name or standard initial quantity in the Name/Std. Qty. (3) For Unknowns, select yes or no in the Calibrator setting. (4) If needed, change the Color. (2) For Standards, enter initial quantity. The number will be displayed as exponents. (3) For a control sample of unknowns, select yes in the Calibrator setting. c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select corresponding wells on the Plate Image and select target on the Plate Image Editor. (2) Select corresponding wells on the Plate Image and select sample on the Plate Image Editor. 17

20 First, select wells Next, select target/sample. After completing the setting, the Plate Image should be like the figure below. [Note] Omit setting Select wells which should be omitted from the analysis and click the Omit button. Those wells will be marked with X like the figure below. Fluorescent data of all of the 96 wells is measured regardless of the omit setting while executing the run. Therefore, if you reset the omit setting, you can use those data for the analysis again. 18

21 (3) Settings of other experiment types There are a variety of setting patterns besides basic Plate Setup described in section (2). Absolute Quantification The setting methods of Absolute Quantification are similar to those of Relative Quantification. Setting example for Absolute Quantification Relative Quantification (Δ ΔCt Method) In the ΔΔCt method, standard samples are not needed to do relative quantification. This method is convenient for high-throughput analysis, because you can use all the wells for unknown samples. Setting example for ΔΔCt method 19

22 Plus/Minus Assay In Plus/Minus Assay, the internal controls can be used to check the possibility of false negative(s). And the data of negative controls are used to set the threshold for the result judgment. a. Open the New Experiment Option window from File to New. b. Select the Plus/Minus Assay Multiplex. c. Internal Control Dye Select the dye to detect the internal control from the pull down menu. d. Making of Target List and Sample List Make the lists by similar methods described before (Refer to section (2)). Sample types are as follows: UNKN (Unknown): Samples to be analyzed. PC (Positive Control) NC (Negative Control) e. Making of Plate Image Set the targets and samples by similar methods described before (Refer to section (2)). And also set the Internal Control (+) / (-) for each well. IC (-): Include the Internal Control IC (+): Not include Internal Control Setting example for Plus/Minus Assay 20

23 SNP Genotyping Assay In the SNP Genotyping Assay, it is necessary to select the filter for Allele1 type and Allele2 type respectively. And the data of negative controls are used to set the threshold for the result judgment. a. Dye setting Select the dye for Allele1 type and Allele2 type respectively from the pull down menu. b. Making of Target List and Sample List Make the lists by similar methods described before (Refer to section (2)). Sample types are as follows: UNKN (Unknown): Samples to be analyzed PC(Allele1): Positive Control of Allele1 type PC(Allele2): Positive Control of Allele2 type NC (Negative Control) c. Making of Plate Image Set the targets and samples by similar methods described before (Refer to section (2)). Setting example for SNP Genotyping Assay 21

24 6.Result/Analysis Analyze data and view graphs in this screen. Using two screens (upper and lower) enables you to evaluate data from various view points. (1) Basic operations a. Click a filter button to display the graph for that filter. FAM and ROX are the standard filters. It is also possible to select multiple filters in the same graph. b. Clicking the Full and Dual buttons switches display from full screen to dual window display mode. c. Select the graph to be displayed from the Analysis Data and Fluorescence menu. d. Select well to be displayed / analyzed in the Selector. Click the Selector tab to show or hide it. e. In dual window display mode, check the box at the top right of the Selector tab to link the selected wells in the two windows. If the check is removed, each one can be selected independently. f. You can choose Well Selector or Target/Sample Type Selector. Well Selector: Select wells to display the graph of those wells. Target/Sample Type Selector: When the sample information has been set in the Plate Setup Screen, it is possible to select all the wells of the same Target or Sample Type by clicking the box. [Note] Using the right-click menu in Well Selector, you can change the kind of sample information displayed and also the omit settings. a g f c b d e Select View > Target. g. You can choose Target, Sample or Sample Type for graph color. 22

25 (2) Graphs and Analysis Amplification Plots Display a curve with fluorescence values on the vertical axis and the number of cycles on the horizontal axis. When the run complete, the optimal parameters are automatically applied and analysis is performed. Verify the analysis results. If the analysis was not performed correctly using the Auto setting, select the parameters manually and perform the analysis again. [Note] If there are multiple targets, the threshold can be set to the optimum position of each target or a position common to all targets. Select either method by checking or clearing the check box of To each target as follows: Individual setting by target Raw: The measured fluorescence value (raw data) Refer to this plot when you need to see the measurement results such as background fluorescence. Common setting to all targets Primary Curve: The primary curve that results from adjustment of the baseline and the last fluorescence value. There are two kinds of threshold for the Primary Curve, horizontal and vertical. Horizontal threshold will be used to calculate the Ct values. Vertical threshold is set to exclude the noisy region from the analysis, in case there are fluorescent fluctuations at the beginning of the cycles. Parameter settings To change the threshold, click the Threshold tab and select Manual. Enter the value in the field on the right or drag the threshold line on the graph. To change the baseline setting, click the Baseline tab and select Manual. You can set the same cycle range for all wells or a different cycle range for each well. Click Apply button after change the settings. 23

26 2nd Derivative: The 2nd Derivative function of the Primary Curve. The Ct value is designated as the maximum point of the 2nd Derivative of the Amplification Plot (SDM: Second Derivative Maximum method). Because this analytical method depends only on the increase rate of fluorescence, it is not necessary to correct baseline and the final fluorescent value. Moreover, it is an excellent analysis method in reproducibility because the Ct value doesn't change as threshold setting changes. Parameter settings The thresholds for 2nd Derivative are set in order to avoid miscalculating the noise level peak as the Ct value. The method of setting the horizontal and vertical threshold is the same as Primary Curve. Analysis Settings ( Smoothing Setting / Normalization ) A smoothing process is performed using a moving average method in order to decrease the fluctuation of measurement and the noise of each cycle. Normalization is to correct the variation in the terminal fluorescent values of amplification plots according to the wells. a. Select [Analysis] > [Analysis Settings] from the menu bar. b. The Analysis Settings window is displayed. c. Smoothing is performed in order to decrease noises in each cycle with the moving average method which averages the variable factors. Select the number of Averaging Points to use for amplification plots and dissociation curves independently. Normalization is set as use default. d. Click the OK button after the settings are complete. Dissociation Curve Display a dissociation curve with fluorescence value on the vertical axis and temperature on the horizontal axis. Raw: The measured fluorescence value (raw data, upper figure). 1st Derivative: The negative derivative of the measured fluorescence values (lower figure). The location at which fluorescence Primer Dimmer 24

27 rapidly decreases is displayed as a peak. The temperature at that time is designated as the Tm value. [Note] In Dissociation Curve Analysis, the temperature of the PCR solution is gradually increased after PCR ends and fluorescence is monitored with SYBR Green. First, the PCR amplification product forms double strands and the SYBR Green I emits fluorescence, but upon reaching a certain constant temperature, it dissociates into single strands and fluorescence decline rapidly. Standard Curve (Absolute Quantification, Relative Quantification) Display a standard curve with Ct values on the vertical axis and the initial template quantity of the standard sample on the horizontal axis. The data of the wells selected on the Selector are plotted on the standard curve. You can select the Ct calculation method form Crossing Point and 2nd Derivative Maximum method. Standard Curve Label Standard Curve numerical formula: Y = A Log(X) + B where Y = Ct value, X = Initial template quantity, and A and B are factors obtained in regression analysis. Coefficient of determination (R2) : It is the second power value of the correlation coefficient, and this value of close to one means high-linearity of the standard curve. PCR Efficiency (Eff) : The PCR Efficiency becomes 100% when PCR products are amplified twice by one cycle according to the theory of PCR. * Mathematical formula to calculate PCR Efficiency from Slope Standard Curve Setting Eff = 10 (-1/Slope) 1 It is possible to use the Standard Curve of previous run for the analysis. a. Click the Set STD Curve button to show the Standard Curve Setting screen. b. Select Manual from D/M list and enter the Slope and Intercept of the standard curve. c. Click Apply button to reanalyze the data. 25

28 Relative Quantity Chart (Relative Quantification) The result of Relative Quantification is displayed in a bar chart. You can select the Ct calculating method from Crossing Point method and 2nd Derivative Maximum method. And also you can select the relative quantification calculation methods Relative Quantity Chart from standard curve method and ΔΔCt method. The bar chart color is Target color only. (Refer to 22 page, g ) Standard Curve Method: This is a general method whereby the Relative Quantities of different samples are obtained by fitting the Ct value of an unknown sample into the standard curve and calculate the initial template quantity. ΔΔCt Method: This is a simple method in which differences in Ct values are converted to Relative Quantities without using a standard curve. In this method, it is a prerequisite that PCR amplification efficiency is close to 100% in all genes. [Note] It is possible to set the standard curve of previous run in Standard Curve Method. This setting is the same with the one in the Standard Curve graph. And these settings will be reflected to each other. Scatter Plot (Plus/Minus Assay, SNP Genotyping Assay) The distribution of data is displayed as Scatter Plot. The data used to judge the plus/minus can be selected from Primary Curve Final, Primary Curve Ct, and 2nd Derivative Ct. The threshold for a plus/minus judgment is set automatically according to the data of Negative Controls. And you can change the threshold value with Manual setting. Plus/Minus Assay SNP Genotyping Assay 26

29 Plate Format (Plus/Minus Assay, SNP Genotyping Assay) This figure displays the judgment result in Plate Format. [Plus/Minus Assay] Plus/minus judgments are done for the Target and the Internal Control respectively. Those results are combined to make an integrated judgment of Plus/Minus for the unknown sample. OK: Controls that reacted correctly OUT: Controls that reacted incorrectly Posi.: Positive Nega.: Negative ND: Not Detected (Both of the Target and Internal Control were not detected.) ERROR: Different judgments in the same replicate Judgment of Target Judgment of IC Integrated judgment [SNP Genotyping Assay] Plus/minus judgments are done for the Allele1 type and the Allele2 type respectively. Those results are combined to make an integrated judgment of Genotype for the unknown sample. OK: Controls that reacted correctly Hetero: Hetero type OUT: Controls that reacted incorrectly Allele1: Allele1 type Allele2: Allele2 type ND: Not Detected (Both of the Allele1 type and Allele2 type were not detected.) ERROR: Different judgments in the same replicate 27

30 Text Report The numeric data such as the Ct and Tm value are displayed in the tabular form. Select the items from the list. If there are many data to show, click the Full button to show the table full screen. Moreover, necessary items can be efficiently selected by appropriately combining the selection of Data Set and Show Items. [Data Set] Data Set of Each Well: Displays the analysis results (Ct value, Tm value, etc.) for each individual well. Data Set of Replicate: Displays the analysis results (mean value, standard deviation, etc.) for each Replicate (a group in which the target and sample setting is the same). Select All: Displays all analysis results. [Show Items] Analysis Setting: Items concerning analytical parameters. CP Method Data: Results obtained from analysis by the Crossing Point method. SDM Method Data: Results obtained from analysis by the 2nd Derivative Maximum method. Items of Data Set of Each Well Well : Well position number Sample Type : Sample Type (UNKN, STD, CAL, NTC, NAC) Target ID : Target (gene) ID Sample ID : Sample ID Filter : Filter used for measurement Ct:: Ct values for individual wells Init Qty : Initial template quantity of the standard sample Qty : Quantity for individual wells Tm#1 : Tm value calculated from the maximum peak of the Dissociation Curve 28

31 Items of Data Set of Replicate (others than above-mentioned) Ct Avg. : Average of replicates of Ct Ct SD : Standard deviation of replicates of Ct Qty Avg. : Average of replicates of Qty Qty SD : Standard deviation of replicates of Qty Rel.Qty : Average of relative quantity of replicates calculated from Ct. Rel.Qty SD : Standard deviation of relative quantity of replicates calculated from Ct. * (CP): calculated using CP method * (SDM): calculated using SDM method [Note] Change the graph settings to display the Chart Properties window. Settings for the X-axis and Y-axis range, scale, and designs of line and symbol can be changed. Double-click on the graph, or select Properties from the right-click menu on the graph. 29

32 7.Export Results (1) Export graphs and data Export each graph Display the graph you want to export and right-click on it. Select the file format from the menu. [Copy] Copy a graph onto the clipboard. After that, you can paste the graph in other application software. [Output Image] Export graph as a bitmap file format. [Export Data] When CSV is selected, the data of graph is exported as a comma separated value file. When Excel is selected, the data and the graph are exported as an Excel file. In the Excel file, you can change the title of the graph or names of samples etc. [Make Report] This software makes a report of your experiment and you can export it as a Word or Power Point file. The report contains basic information of the experiment, such as file names, so it's convenient for data management. [Print] When you select Print, the preview screen will be displayed. You can print this screen or save it as a PDF file. 30

33 Export Data It is possible to export data of Text Report as a CSV file or Excel file. From the right-clicking short cut menu, select [Export Data] > [CSV] or [Excel]. Make Full Report Make a full report with some figures. Select [File] > [Make Full Report] to show the Report Setting window. Check the graphs you want to report and select the file format. Click OK and the report will be created. Example of report: Power Point 31

34 (2) Export files for RDML Select files you need for making of RDML file, and export them. a. If needed, change the Run ID. b. Select Detection Method, CP or SDM. c. Select files you want to export. d. If needed, select items to be exported. e. Click Output button to export the files. d. Select items by each check box. c. Select files by each check box. 32

35 8.Appendix (1) Consumption 48 well plate Cat.# Product Quantity Comments NJ well Snap Plate 20 plates Can be separated into 2 pcs of 24-well plate NJ720 Flat Cap for Snap Plate 120 strips Cap for NJ700 Flat cap for snap plate (Cat.# NJ720) Strip Tubes 48 well snap plate(cat.# NJ700) Cat.# Product Quantity Comments NJ ml Hi-8-Tube 125 strips Strip of 8 attached caps for use with 0.2 ml strip tubes NJ ml Hi-8-Flat Cap 125 strips Flat caps for easy closure and sample labeling 0.2 ml Hi-8-Flat Cap(Cat.# NJ302) 0.2 ml Hi-8-Tube(Cat.#NJ300) Strip Tube with individual Flat Cap Cat.# Product Quantity Comments NJ ml 8-Strip Tube, individual Flat Caps 125 strips Strip of 8 tubes with each cap. Can use one tube by separation. 0.2ml 8-strip tube, individual Flat Caps(Cat.# NJ600) 33

36 (2) How to use 48 well plate and strip tube a. Prepare the PCR reaction plate or tubes. b. Close the cap firmly. c. Briefly centrifuge the plate or tube to put down the solution at the bottom of the well and remove air bubbles. Appropriate Inappropriate Reaction solution positioned at the bottom of well Reaction solution on side wall of well Air bubble at bottom of well d. Set the tubes or plate in the sample block. If you use only few tubes, set the reaction tubes at the center of the sample block and put empty tubes at the both side. [ Caution ] Do not touch the plate or 8 strip tube with bare hands. This may cause fluorescence noise. Use only flat caps. Don't use dome caps.! Warning The temperature of the lid rises to 100 C or more during operation, and there is a danger of bodily injury from burns if it comes into contact with skin. Also, after the run is completed, the sample block may remain for some time at the final set temperature. In either case, take care not to touch hot parts of the equipment. 34

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