Qiagen QIAcuity 911000 User Manual page 204

Application
a) Images or analysis data cannot
be viewed
b) Poor or no amplification
c) No clear separation between
positive and negative partitions
d) Images are saturated
e) Sample result is 0 copies/ µl or
infinite in absolute quantification
f) Sample results of replicates differ
a lot
g) High copy number in NTC
h) Lower RFU of negative partitions
in NTC / samples with low
number of positive partitions
i) The confidence interval is wide
j) Vertical stripes in the images
QIAcuity User Manual 07/2020
Comments and suggestions
Check the connection to the QIAcuity instrument.
Check if the correct protocols and reagents have been used.
Check if the reaction was set up correctly.
Check the cycling and imaging conditions.
Check the starting quality and quantity of the template. We
recommend that you use QIAGEN kits for sample preparation.
Check if the correct protocols and reagents have been used.
Check if the reaction was set up correctly.
Check the cycling and imaging conditions.
Check the starting quality and quantity of the template. We
recommend that you use QIAGEN kits for sample preparation.
Re-image the plate with 30% lower exposure duration or lower gain
settings (see also section Image quality control)
If your concentration is 0 copies/ µl, although the sample is not an
NTC, check the Histogram or 1D Scatterplot for this well. In case you
have nearly only positive partitions in the well, a proper auto-
threshold setting was likely not possible. Please check also if the
image of the well is too dark, and in case re-image the plate with
30% higher exposure time or gain settings.
Check the images for blacked-out areas, that can occur e.g. due to
bad filling or areas of low amplification (see section Image
corrective measures)
Check the images for dust or other particles. In case, wipe the plate
with a lint-free tissue (optionally, use ethanol) and re-image the plate.
The signal intensity might be lower in images with high number of
negative partitions. There's no influence on the result analysis, as the
signal to noise is not affected.
The number of valid partitions is low. Check the images for blacked-
out areas, that can occur e.g. due to bad filling or areas of low
amplification (see section Image corrective measures)
Please re-image the plate for proper image analysis
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