PNAClamp Analyzer (Version 1.0.2) User Manual

PNAClampTM Analyzer
(Version 1.0.2)
User Manual
PNAClamp
TM
TM
Analyzer, a program supplied to the customers of PNAClamp
Mutation Detection kits
just as a service program, makes analysis of PNAClamp experiment data from several Real-Time
PCR systems much easier and faster.
Contents:
TM
Part I. What is the PNAClamp
Analyzer? ...................................................................................................................... 2
1. What is this program? .................................................................................................................................................. 2
2. Why is this program useful? ...................................................................................................................................... 2
TM
3. Which PNAClamp
Mutation Detection kits are compatible with this program? ............................... 2
4. Which realtime PCR instruments are compatible with this program? ..................................................... 3
5. What are the restrictions to use this program? ................................................................................................. 3
Part II. How to use this Program? ......................................................................................................................................... 4
1. Installation ......................................................................................................................................................................... 4
2. General procedure to use this program ............................................................................................................... 4
TM
3. An example to use this program with PNAClamp
EGFR Mutation Detection kit. ....................... 10
Instruction Version : Ver.1.0.0
Date of Revision : 2012-May
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PNAClamp Analyzer
User Manual Version 1.0.0
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Part I. What is the PNAClampTM Analyzer?
1. What is this program?
TM
The PNAClamp
TM
PNAClamp
Analyzer is a simple program that can interpret the results of several
Mutation Detection kits as easily understandable and manipulatable form.
This program is not for sale:
-
Panagene provides this program without any cost to the customer of PNAClamp
TM
Mutation
Detection kits who use the Bio-Rad’s CFX®96 realtime PCR system in the purpose to serve
convenience of using the kits.
-
This program will interpret the result with the basis of analysis criteria described in the
instruction manuals of the kits. In other word, this program will do the simple calculations
and type the result instead of user.
-
The accuracy of the result depends only upon the kit, instrument, and user.
Therefore, this program has no responsibility to the result.
2. Why is this program useful?
TM
The PNAClamp
Mutation Detection kits are one of the most powerful one among molecular
diagnostics products in the aspects of accuracy and sensitivity. However, they have dependency to
realtime PCR instrument used and also give the results from the combination of data from separate
reactions in the view of the instruments. Even though the results can be interpreted with simple
correlations, but user should repeat boring work to get them. This program would serve the boring
work in seconds for each result and give time to do more important things for user.
3. Which PNAClamp
The PNAClamp
TM
TM
Mutation Detection kits are compatible with this program?
TM
Analyzer is compatible with four (4) PNAClamp
Mutation Detection kits listed
below:
-
TM
BRAF Mutation Detection kit
TM
EGFR Mutation Detection kit
TM
KRAS Mutation Detection kit
TM
PIK3CA Mutation Detection kit
PNAClamp
PNAClamp
PNAClamp
PNAClamp
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PNAClamp Analyzer
User Manual Version 1.0.0
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4. Which realtime PCR instruments are compatible with this program?
TM
The PNAClamp
-
Analyzer is compatible with realtime PCR instruments listed below:
Bio-Rad : CFX96®
5. What are the restrictions to use this program?
A. Computer System Requirements.
-
Supported Operating System:
Microsoft Windows XP with Service Pack 3 or higher, Microsoft Windows Vista, and
Microsoft Windows 7 (except starter version).
-
Microsoft Software:
· Microsoft Office 2007 or higher, or Microsoft Excel 2007 or higher
B. Operational Requirements. (Please see next chapter for detail procedure and rules)
-
The clamping controls and every reaction mixtures should be placed with the proper order in
the 96 well plate (or the block in case of using use the 8 strip tubes) equipped to the realtime
PCR instruments.
-
After analysis of Ct (or Cq), the all data should be exported to excel file(s) with the “Export to
Excel File” function of the Operating/Analysis program of the used realtime PCR instrument.
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User Manual Version 1.0.0
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Part II. How to use this Program?
1. Installation
This program is distributed as a standard executable setup file. Installation will be started by just
double-clicking the Setup file in the windows explorer. All the installation procedure is familiar enough
to finish the installation without see any document.
2. General procedure to use this program
A. Preparation rule of the 96 well plate to be equipped into the realtime PCR instrument.
-
Be Careful!
It is very important to position the clamping controls and each reaction mixture
of each sample correctly because there is no way to identify the each reaction by the
program itself.
-
Therefore, wrong positioning means wrong results directly.
Please see the following figures (Figure 1A~1D) for the examples of the rules for positioning.
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Figure 1A. The examples of the rules for positioning of clamping controls and each reaction mixtures.
TM
(PNAClamp
BRAF Mutation Detection Kit)
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Figure 1B. The examples of the rules for positioning of clamping controls and each reaction mixtures.
TM
(PNAClamp
EGFR Mutation Detection Kit)
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User Manual Version 1.0.0
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Figure 1C. The examples of the rules for positioning of clamping controls and each reaction mixtures.
TM
(PNAClamp
KRAS Mutation Detection Kit)
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User Manual Version 1.0.0
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Figure 1D. The examples of the rules for positioning of clamping controls and each reaction mixtures.
TM
(PNAClamp
PIK3CA Mutation Detection Kit)
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B. Operation and First Analysis to get Ct data.
-
The description about these steps is skipped because they will not affect to the functionality
TM
of this program. Please see the instruction manuals of the PNAClamp
Mutation Detection
kits and of the realtime PCR instrument provided by manufacturer of the instrument.
C. How to export data to excel file(s) from the realtime PCR instrument?
-
Bio-Rad’s CFX96®:
Click “Export All Data Sheets to Excel” in “Tools” menu as shown in Figure 2A.
(Bio-Rad CFX Manager Ver.2.0)
Figure 2A. The position of “Export All Data Sheets to Excel” menu
in the data analysis program of Bio-Rad’s CFX96®.
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3. An example to use this program with PNAClamp
TM
EGFR Mutation Detection kit.
1) Execute the program:
Window Start Menu> Programs> Panagene> PNAClamp Analyzer (shortcut)
[Home Screen of the program]
2) Start new analysis:
[Program Menu] File > “New Analysis”
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3) Select analysis method:
Select appropriate an “Analysis Method” in the popup window, and then click “OK” button.
4) Open excel data file to be analyzed:
4-1) Select the manufacturer’s name of the real-time PCR instrument used in the “File Type” option of
the “Open” window.
4-2) Find the exported excel file to be analyzed.
4-3) Click the “Open” button of the “open” window.
4-4) Analysis of data will be done automatically.
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5) Check the analysis result:
Map area : The location and test result can be checked briefly as like the 96-well plate image.
Yellow: Clamping control
Blue: Wild type
Red: Mutant type
Grey: Invalid data (the test result could not be determined)
Data area : The Ct value, ΔCt value, and the test result will be shown.
1. If the cell color of ΔCt-1 is red, the sample is assessed as mutant. (ΔCt-1 value over 2.0)
2. If the cell color of ΔCt-1 is green and the cell color of ΔCt-2 is red, the sample is assessed as
mutant. (ΔCt-1 value in 0 to 2.0 range & ΔCt-2 value below 3.0)
- Sample information (sample ID, information, and comment) can be added or edited.
Graph area : When a sample is selected in the Map area or in the Data area, the corresponding
amplification plot of the sample will be displayed in the Graph area. At the same time, the
corresponding wells in map area will turn into blue.
TM
** When open the saved data file, please open it after run the PNAClamp
Analyzer.
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6) Supported Options
- Data Menu: Copy data (either only selected or all data)
- Graph Menu: Currently displayed graph can be copied and saved as an image file. All graphs of the
samples can be also saved at once as corresponding image files.
- Report Menu: Display final report and/or print the report
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- When click “Report Preview”, following screen will be displayed (When the name, sex, birthday,
and/or comment of the sample is added/or edited previously using this program, it will be shown on
the report as well)
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