Esco Swift Real-Time Thermal Cycler. Absolute Quantification User guide

Transcription

1 Esco Swift Real-Time Thermal Cycler Absolute Quantification User guide

2 Prepare all materials needed Extracted DNA sample Controls DNA polymerase Dyes/probes Internal controls PCR buffer dntps Primers Standards Samples such as blood or tissue cannot be used for PCR. DNA should first be extracted from these. Refer to the kit manufacturer instructions on how to extract DNA from these samples. It is good methodology to use controls. For negative control, PCR grade water is used. Other controls such as positive controls (DNA from known positive sources) and other negative controls (DNA from known negative sources). Master Mix components + Standards (Usually included in the kit if a kit is used) Always plan ahead

3 Select Real-Time PCR application Absolute quantification Uses standards with known concentrations to quantify unknown samples Relative quantification Uses house keeping genes to estimate expression levels SNP analysis SNP: single nucleotide polymorphism Click to enter Absolute Quantification

4 ? Which application should I select? Answer: When in doubt, ask the manufacturer of the kit you are using. A quick answer would be that, if standards are used (i.e., they are included in the kit contents), then it is very likely that you should select Absolute Quantification. Most diagnostic test that are done via Real-Time PCR are done with Absolute Quantification, e.g., -HBV -TB -Etc.

5 Interface 2 3 Enter user and test details 2 3 Enter tube volume Enter temperature cycle details

6 Example thermal cycle parameters PCR cycles: 40 cycles 37 C for 5 mins 94 C for 2 mins 95 C for 5 secs 60 C for 40 secs * WARNING: The values shown here are for illustration purposes only and would vary between different test kits and manufacturers. *-Detect fluorescent signal at this step

7 Enter the parameters into the interface PCR cycles: 2 40 cycles 37 C for 5 mins 94 C for 2 mins 95 C for 5 secs 60 C for 40 secs * *-Detect fluorescent signal at this step Click Cycle/Temp to set a program cycle 2 Enter target temperature and time. Leave the rest at default. Take note that a graph representing the program being set up is shown.

8 Enter the parameters into the interface PCR cycles: 2 40 cycles 37 C for 5 mins 94 C for 2 mins 95 C for 5 secs 60 C for 40 secs * *-Detect fluorescent signal at this step Click Add to add another step in the program cycle. 2 Enter target temperature and time. Leave the rest at default. Take note that the cycle number is set to. This means that the thermal cycler will run through the steps in the program cycle only once.

9 Enter the parameters into the interface PCR cycles: 2 40 cycles 37 C for 5 mins 94 C for 2 mins 95 C for 5 secs 60 C for 40 secs * *-Detect fluorescent signal at this step Click Cycle/Temp to set another program cycle. Another program cycle is needed because we need to set next steps to cycle for 40 times (instead of just once) 2 Enter target temperature and time. Leave the rest at default.

10 Enter the parameters into the interface PCR cycles: 2 40 cycles 37 C for 5 mins 94 C for 2 mins 95 C for 5 secs 60 C for 40 secs * *-Detect fluorescent signal at this step Click Add to add another step in the program cycle. 2 Enter target temperature and time. Leave the rest at default. IMPORTANT: Because we want to detect for the fluorescent signals at this step, set sample mode to Single. 3 DO NOT FORGET to set the number of cycles to the desired amount!

11 Enter the parameters into the interface PCR cycles: 40 cycles 37 C for 5 mins 94 C for 2 mins 95 C for 5 secs 60 C for 40 secs * *-Detect fluorescent signal at this step Click End to end the thermal cycle programs. 2 Select SOAK to set the machine to keep the samples at 8 C (recommended) 3 REVIEW the programs! Check that everything is entered correctly Temperatures Times Number of cycles

12 Enter sample data Click the Sample Data tab to go to the next interface screen.

13 Enter sample data In this example, 8 wells are selected. Set the sample information for each tube in the corresponding well. Multiple wells can be selected to make the setting sample information easier and faster. To select multiple wells, you may click and drag and/or Ctrl+click

14 In this example SYBR (SYBR Green Dye) is used. Enter sample data The setting (dye selection) is set for all highlighted wells. 2 Select which dyes will be used. Up to 8 dyes/channels can be used simultaneously. 2 Click Add to set the dye.

15 Enter sample data Standards 2 The setting (standard) is set for all highlighted wells. In this example, 5 standards are placed in the first 5 wells. Select which wells contain the standards. 2 Click the Std box to set the wells as standards.

16 STD 8,000,000,000 copies/ml Enter sample data Standards The standard in the first well is selected and is indicated to contain 8,000,000,000 copies/ml. 2 IMPORTANT: The concentration of each standard should be set. IMPORTANT: Highlight the particular standard you want to set. Make sure to select them one by one. 2 Enter the concentration value. DO NOT FORGET to also indicate the units used.

17 Enter sample data Standards STD2 800,000,000 copies/ml STD3 80,000,000 copies/ml STD4 8,000,000 copies/ml STD5 800,000 copies/ml Repeat for all standards. In this example, the first 5 wells contain the standards.

18 Enter sample data Unknowns 2 The setting (unknown) is set for all highlighted wells. In this example, 2 unknowns are placed in the 6th and 7th wells. Select which wells contain the unknown samples. 2 Click the Unk box to set the wells as unknowns.

19 Enter sample data Controls 2 The setting (negative control) is set for all highlighted wells. In this example, nontemplate control is placed in the last well. Select which wells contain the controls samples. 2 Click the Neg box to set the well as negative control.

20 Enter sample data! The samples may be named/labeled accordingly by entering the names in the appropriate field. Other data may also be entered such as Sex, Age, and other sample details if applicable.! Be sure to enter the names of each tube/well one by one. If multiple wells are selected/highlighted, the changes will be done for all selected wells and previous settings might be overwritten.

21 2 Run the programs Fluorescence detection of signals can be monitored in real time here The temperature cycles can be monitored in real time here Click the Run Programs tab to go to the next interface screen. 2 Click the (!) button to run the program.

22 Analysis of data

23 Example of run data

24 Here we can see that the 5 standard concentrations are highlighted (blue) Analysis steps Important: Highlight the concentration values of the standards that you want to use. To highlight them, use Ctrl+click.

25 Analysis steps 2 Choose which analysis method will be used. 2 Click through the available analysis steps. (Setting of the Baseline is not available if 2nd Deriv. Max. is chosen.)

26 ? Which method should I use? Amplification curve () st derivative (2) 2nd derivative Answer: Either 2nd Derivative Maximum method or Fit Points method can be used to obtain the calculations. The difference is the mathematical principle used in calculating Ct values.

27 For the following slides, Fit Points method is used Analysis steps FIT POINTS Zero Adjust Click Zero Adjust. 2 Select Auto then click OK. Zero will be adjusted to the average from cycle to 0. This can be manually set if desired.

28 ? What is Zero? Fluorescence Zero level 0 0 Cycles Setting the zero level sets the point at which the level of fluorescence is measured as zero. Selecting Auto sets the zero level from the average from cycle to 0.

29 Analysis steps FIT POINTS Zero Adjust Note that the zero level for fluorescence has been adjusted.

30 Analysis steps FIT POINTS Baseline 2 Click Baseline. 2 Adjust the baseline (red horizontal line) to be as low as possible but above the noise. To adjust, you may click and drag it or a value may be entered manually in the appropriate box.

31 ? What is Noise? Fluorescence Baseline Zero level 0 Noise Cycles The amount of fluorescence due to amplified DNA at the first few cycles is expected to be very low and less than the lower limit of detection of the detector. Thus any signals that are found here are typically considered random/erratic noise signals and should not be included in the analysis.

32 Analysis steps FIT POINTS Analysis 2 Click Analysis. 2 Adjust the threshold (red horizontal line). The threshold is the basis for determining the Ct values. Ideally, the threshold line should be at the linear-log phase of the amplification curve. Usually, this can be set to 0x the Baseline SD. To adjust, you may click and drag it or a value may be entered manually in the appropriate box.

33 ? What is Threshold? Threshold Fluorescence Log-linear phase Plateau phase Baseline Zero level 0 Early exponential phase Linear ground phase Phases of the PCR amplification curve Cycles The threshold is the basis for determining the Ct values. Ideally, the threshold line should be at the linear-log phase of the amplification curve. Usually, this can be set to 0x the Baseline SD.

34 ? What is Ct? Threshold Fluorescence Baseline Zero level 0 Ct Cycles The Ct (cycle threshold) value is determined by the intersection of the amplification curve with the straight line obtained by sample fit.

35 ? How is concentration calculated from Ct? STD STD2 STD3 STD4 STD5 Ct value of the unknown sample Calculated concentration for unknown sample The Ct values of the standards with known concentration are plotted in a graph (as shown above) and regressed to determine a straight line. The concentration of the unknown samples are determined by tracing their Ct values in the line graph.

36 Interpreting Real-Time PCR data

37 What data do you get at the end? Ct values Calculated concentrations The Ct values and calculated concentration can be found here

38 ? How do I know if the test resulted positive or negative? Generally, if a test kit is used, the kit manufacturer should provide the criteria to determine whether the results of a PCR run is positive or negative.

39 ? How do I know if the test resulted positive or negative? For example, the Liferiver HBV Quantitative Real Time PCR Kit indicates that: Sample Ct value (FAM) Sample Ct value (HEX) Interpretation 38 Signal detected POSITIVE Signal detected Repeat run again and if Ct value is still 38-40, then NEGATIVE No signal detected Signal detected NEGATIVE No signal detected No signal detected No diagnostic statement can be made. (The PCR reaction did not run properly or was inhibited)

40 End