Techne PrimeQ User Manual download pdf (Page 106)

106

The crossing line is a best-fit of where the reaction efficiency is at its highest and most constant

for each reaction curve. The quantification cycle, or Cq, (sometimes referred to as the C

in the

literature), is the point at which an amplification curve intercepts the crossing line. Measuring

fluorescence at the Cq for each well will allow the most reliable well-to-well comparison.

Plotting the Cq against the known concentration of standards will generate a standard curve that

makes it possible to estimate the starting concentration of unknowns.

The quantification cycle (Cq) is the point

where the amplification curve intercepts the

crossing line.

Two approaches are available for calculating the quantification cycle on PrimeQ: fit points and first

derivative maximum.

3.9.3 Fit points

This approach is the preferred approach when using low copy number templates and is based on

the setting of a crossing line and noise threshold. It is the default method used by Quansoft. The

noise threshold is used to define all non-informative fluorescence data – data that is typically

indistinguishable from the fluorescent noise seen in the early stages of amplification.

The crossing line is set at a specified number of standard deviations (SD) above the noise line

using either the default settings or those specified by the user. The default setting is 10 standard

deviations above the noise, which is the average of fluorescence measured in cycles 3–10.

Fluorescence is then plotted on a log scale vs. cycle number with the two cursors placed in the

appropriate positions: a blue line showing the noise threshold and a red, the crossing line.

The quantification cycle is then calculated using fit points. These are a defined number of

fluorescence points situated on the curve above the crossing line (default number is 2). The

software uses these points in a linear regression calculation to determine the best-fit gradient for

each well. The intersection of this line through the crossing line is the Cq and this will be a

fractional cycle number. The Cq is calculated for each well.

Note on log scale: The only real difference between a log and linear scale is the way the data is

displayed. The lower values in the log scale are ‘blown up’ to make the data more readable such

that the normalized values approaching zero can be seen more clearly. The small increase seen in

Cycle Number

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Relative Fluorescence (Log)

0.01

0.1

Crossing line

Noise threshold

Cycle Number

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Relative Fluorescence (Log)

0.01

0.1

Crossing line

Noise threshold

Cycle Number

45403530252015105

Relative Fluorescence (Log)

0.1

High copy

numbers

Low copy

numbers

Cycle Number

45403530252015105

Relative Fluorescence (Log)

0.1

High copy

numbers

Low copy

numbers

Cycle Number

45403530252015105

Relative Fluorescence (Log)

0.1

High copy

numbers

Low copy

numbers

Cycle NumberCycle Number

45403530252015105

Relative Fluorescence (Log)

0.1

High copy

numbers

Low copy

numbers

Cycle Number

45403530252015105

Relative Fluorescence (Log)

0.1

High copy

numbers

Low copy

numbers

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