Setting up a Run of Real-time PCR CFX 96 Real-Time System

Transcription

1 Version: 1.0 Last Revision Date: Setting up a Run of Real-time PCR CFX 96 Real-Time System (BioRad) generi biotech

2 OBSAH 1. Setup of already existing or setting of a new temperature profile Setup of already existing temperature profile Setting of a new temperature profile Ct values analysis Allelic discrimination assessment...21 generi biotech 2/24

3 1. Setup of already existing or a new temperature profile 1.1. Setup of already existing temperature profile Quickly spin a CFX plate with samples Put the plate into a cycler (make sure that the A1 well is oriented into the upper left corner) Click the Bio-Rad CFX Manager icon to run the software (2.0 version). When using updates (e.g version) select the run type User-defined. generi biotech 3/24

4 Select the Create a new Run option (2.0 version). generi biotech 4/24

5 In the Protocol tab click the Select Existing button. Select the suitable temperature profile in the menu and open it. You can also choose the profile from Express Load option. The title of the selected profile appears in the Selected Protocol frame. Note: Prior to use of the Express Load option it is necessary to save temperature profiles to folder Express Load in the PC. In the Preview frame you can check the temperature settings, plate scanning, sample volume and number of repeats. generi biotech 5/24

6 Click the Next button. In the Plate tab you can check the plate layout and scanned fluorescent channels ( Fluorophores ). generi biotech 6/24

7 Click the Next button. In the Start Run tab click the Start Run button. Determine the file name and folder, where the PCR outcomes will be saved. The run starts after clicking the Start Run button and then Save. generi biotech 7/24

8 1.2. Setting of a new temperature profile Quickly spin a CFX plate with samples Put the plate into a cycler (make sure that the A1 well is oriented into the upper left corner) Click the Bio-Rad CFX Manager icon to run the software and select a new run Create a new Run (2.0 version). When using updates (e.g. 3.1 version) select the type of the run User-defined. generi biotech 8/24

9 Startup Wizard allows you to set up the temperature profile In the tab Protocol select Create New generi biotech 9/24

10 Set up the temperature profile in the Protocol Editor new window. First set the reaction volume (usually 20 or 25 µl). Perform changes with double click on temperature and/or duration time values in the blue background. It is possible to set a number of repeats. E.g. if you need the section labeled with red arrow to be performed 40, it is necessary to set the number of repeats 39 (i.e. how many times it will be repeated).. In the lower left part of the window you can adjust the temperature profile through Inserting of a step Inserting of a temperature gradient (see next chapter) Inserting of repeats of particular steps Inserting of melting curves Adding another plate scan Setting of temperature and duration time of each step Deleting a step generi biotech 10/24

11 Setting of a temperature gradient Clicking the button Insert Gradient allows you to insert a temperature gradient into a temperature profile. This is done through double clicking on a temperature value in the dark blue background. Set maximum and minimum temperatures. The gradient in detail is displayed in the lower right part of the window Click on the OK button and save the file with new temperature parameters. generi biotech 11/24

12 In the Plate tab click on Create New again. In the Plate Editor - New you can set the plate layout, acquisition in fluorescent channels, groups of samples etc. Clicking the button Select Fluorophores allows you to select the fluorescent channels to be scanned. generi biotech 12/24

13 After clicking the OK button save the file with your settings. The title of the file appears in the Express Load menu with each new run. generi biotech 13/24

14 Click on Next, then click on the Start Run button in the Start Run tab. Determine the file name and folder, where the PCR outcomes will be saved. The run starts after clicking the Start Run button and then Save. After the run starts you can display time remaining to the end of the run in the tab Time Status. generi biotech 14/24

15 2. Ct values analysis 2.1. Launch the Bio-Rad CFX Manager program on your computer and open the required file using the open folder icon In the Quantification tab select the scanned fluorescent channels, and mark Log Scale if you want to convert the values to logarithmic scale (recommended). generi biotech 15/24

16 2.3. Next it is necessary to set up the threshold value (optimally in the middle of the linear part of the logarithmic curves). In the tab Settings Baseline Threshold the auto calculated threshold value is displayed, you can adjust the value according to your requiremnts ( User Defined ). generi biotech 16/24

17 In case of inappropriate values of Baseline Cycles for some of the curves, it is possible to adjust the range manually in the fields Begin and End (in User Defined mode). generi biotech 17/24

18 2.4. Using the icon Trace Styles you can mark the samples with colors. First select the required samples then choose a color in the frame Selected Wells. This way you can distinguish between e.g. NTC, standards and groups of samples It is possible to exclude wells from analysis through clicking on the respective wells (gray color), Ct values will not be displayed. The rest of the plate remains blue. generi biotech 18/24

19 2.6. In Settings Analysis Mode menu select Baseline Subtracted Curve Fit (version 2.0). In Settings Baseline Setting menu select Baseline Subtracted Curve Fit (version 3.1) The tab Quantification Data - Results displays: Position of a well ( Well ) Analysed fluorophore ( Fluor ) Ct values ( Threshold Cycle ) generi biotech 19/24

20 It is possible to order values through clicking on the title of a column (green arrow in the picture) or arrange columns next to each other. Note: You can turn off the function of curve highlighting using your mouse in Settings Mouse Highlighting Other tabs Run Information The tab includes parameters of the finished run: temperature profile and its title, date and time of the run or e.g. sample volume. Gene Expression End Point Allelic Discrimination (see chapter 3) The data for allelic discrimination must be obtained from multiplex reaction including at least two fluorophores. Using one fluorophore enables the identification of one type of allele in the sample. generi biotech 20/24

21 3. Allelic discrimination assessment 3.1. After launching a program (through double clicking on.pcrd file) remove the fluorescent channels which you do not want to evaluate. For allelic discrimination analysis two different fluorescent channels (usually FAM/Sybr and HEX/JOE) are used. generi biotech 21/24

22 3.2. In the tab Allelic Discrimination all of the samples are distributed according to their genotypes. In the upper left window you can see a chart of RFU ( relative fluorescence units ) values with x axis for allele 1 and y axis for allele 2. A group of heterozygote samples is situated between the homozygote ones. There is a tab in the upper right window with resulting genotypes for particular samples (column Call ) including RFU values. Note: It is convenient to remove empty plate wells from analysis (grey color). Their genotypes will not be displayed anymore. generi biotech 22/24

23 3.3. Another method of displaying genotypes graphically is available through checking the Polar Coordinates box. X axis represents the angle of a sample position with x axis from preview depiction (version 3.1). generi biotech 23/24

24 3.4. After checking the View call map box, each well of a plate is colored according to its genotype (here: orange WT, green HET, blue MUT). generi biotech 24/24