affigene Software
Q: I am not able to import the text file from my run. The software reports "A calculation-error occurred: Object reference not set to an instance of an object. No report is available" why?
A: The unknowns may not be defined with both FAM and ROX. To be able to import the text file all unknowns must have a value for both FAM and ROX. Deselect the unknown wells which do not have both dyes defined before exporting the raw data - and the affigene® analysis software will accept the text file.
Q: Is it possible to download data from the Mx3000P instrument after shutting down the instrument and/or the computer?
A: Yes the latest performed run can always be downloaded even if the instrument and/or the computer has been shut down and started again.
Q: The test is not approved in the affigene® analysis software because of failed NTC (non-template control). Can I get a print-out of the results for the negative samples, provided the IC is valid for those samples?
A: This is not possible to do in the affigene® analysis software where we follow the IVD requirements for CE-labelled products. You can however get a print-out of the Ct-values even if the test is not approved. All Ct-values are displayed in the end of the report.
Q: Why do not all amplification-curves reach the same end fluorescence (dR Last) in the same run?
A: One advantage of real time PCR compared to conventional PCR is that in real-time PCR one measures the Ct value, which is found in the logarithmic phase of the amplification rather than at the end fluorescence (conventional PCR / end point detection). The variability is significantly lower in the logarithmic phase.
Q: I got an error message saying "Test NOT approved" and "Result is not reported as test is not approved". What is wrong?
A: First of all, check the software to see what has failed:
Criteria not met:
a.) IC check
There is a control algorithm to the sample extraction parameters which checks validity of the entered values in relation to the IC volume. If you receive this kind of error there are two options, either you have:
a.) followed the protocol in the User Manual, but entered an incorrect value by mistake. Enter the correct value and reanalyze the results only.
b.) not followed the protocol in the User Manual, therefore the entered combination of values are not valid. This run is not reliable! The sample preparation should be repeated.
b.) NTC Test/ Negative control test
This means that either the Negative Template Control is positive (i.e it has been contaminated), or the Negative Template Control has no amplified Internal Control (i.e it has been inhibited). This run is not reliable! The sample preparation should be repeated.
c.) Threshold Check
This means that the threshold-level is not within criteria. This run is not reliable! Check expiry date of the amplification kit.
d.) FAM Ct for target trender Low/ Positive control test
This means that the lowest calibrator/ positive control is not within criteria. This run is not reliable! The amplification should be repeated.
Q: I got a message saying "Re-run sample and check manual for more info!" What does that mean?
A: There can be different reasons, see a-d below:
a) This means that the sample has been inhibited in terms of amplification. The sample preparation should be repeated. Potential inhibitors are for example; triglycerides, haemoglobin, bilirubin, protein or genomic DNA.
b) inefficient amplification; there is an amplification curve but the fluorescence is too low.
The sample preparation should be repeated.
c) a drifting curve without any amplification; check the curve in the Mx-instrument-software. This sample could be false positive and the amplification should be repeated.
d) a sample with an extremely high titer-level; check the curve in the Mx-instrument-software. This sample should be diluted before sample preparation is repeated.
Q: How can I check the life-time of the lamp in the Mx3000P instrument?
A: Go to "Lamp Reset and Utilities" in the Mx3000P instrument program menu. A dialog frame will appear and show the total time of the lamp. The average life expectancy of the lamp is 2000 hours.
Q: I get an error message saying "A calculation-error occurred: No NTC-well found No report is available". What is wrong?
A: The NTC-well is not defined correctly in the setup, the NTC-well should be defined with the assay name i. e for CMV: "CMV trender NTC".
Q: I got a bad run with almost no amplification at all and looking at the PCR tubes afterwards most of the liquid is stack up in the lids, what went wrong?
A: This could be caused by:
a) wrongly used plastic ware not suitable for the Mx3000P
b) tubes not tightly closed
c) the heated lid which has failed to heat. To check this, run a plate with water over 40 cycles and see if you still have evaporation all over the plate. In that case the heated lid has failed to heat.
Q: The software reports a titer and a message ***. What does that mean?
A: This is reported for the affigene® tracer assays and means that it is a positive sample in the range of LOD. In the range of LOD, a sample that is run in many replicates may not be determined as positive for all replicates.
Q: What is the "baseline"?
A: The baseline is the level where no product is detected as the fluorescent signal is below the detection limit of the instrument. The baseline is the extension of the line set from the first temperature cycles.
Q: The test passes the criteria and all titers are calculated, but there is an error message ("Not as specified") along with the parameters. What is wrong?
A: This is the threshold warning which indicates that the threshold level in the instrument is not set according to specification. This may affect the slope of the standard curve which will affect the titer calculations. The further away from the recommended setting, the greater the deviation is in the titer-calculations.
Note: With some instruments it may be impossible to set the specified value, in that case set a value as close as possible.
Q: Why can't I export the results?
A: You must enter information in all header fields, such as: Operator, Date, Kit lot# and test ID before any data can be exported.
Q: Why can't I print the results?
A: You must enter information in all header fields, such as: Operator, Date, Kit lot# and test ID before any data can be printed.
Q: Occasionally I get a message saying "Several testtypes found in data, please load data from one test at a time". What does that mean?
A: The software can only handle one assay at a time due to different assay criteria, please make sure that each file only contains data from one assay type. If you only have performed one assay this message will also turn up if you do not define the negative control and the calibrators correct. Example: The negative control is defined as "NTC" instead of "CMV trender NTC". If the negative control and the calibrators are wrongly defined the software will think that there are different assays.
Q: I couldn't set the threshold because there was no IC signal for the NTC
A: a) No IC signal is caused by inhibition ("rerun" in the software).
b) The IC was not added during the extraction procedure.
c) ROX is not selected in the software.
d) The NTC is contaminated (very much contaminated). In competition with the contaminating DNA the IC gives no sign
Q: Why do you assign a fixed Ct value for calibrator High?
A: The known concentration of calibrator High corresponds to a fixed Ct-value. The two calibrators Low and High create the standard curve. Calibrator Low must be within the specific interval for the assay.
Q: What is the "threshold"?
A: The threshold is the level set where the fluorescence signals are significantly above the background signal. The threshold level is set utilising the trender calibrator High or the tracer PC amplification plot.
Q: I didn't get amplification in any well.
A: Most likely the samples are put in other wells than those defined in the MX-software. Note: When importing earlier set-up protocols ensure that you put the samples in the same rows and that you have defined the correct number of samples.