Techne PrimeQ Relative Quantification Analysis

PrimeQHelp@bibby

-

scientific.com

| www.bibby

-

scientific.com

[phone]

1

Technical Note T08-002A

Figure 1

:

The

Results Editor

home screen.

Relative Quantification Analysis

General Introduction to Data Analysis

The aim of this Technical Note is to explain the principle of relative quantification analysis and to guide you through
the experimental set up and analysis of data. It begins with a general introduction to data analysis.

Results Editor

The starting point for data analysis is the Results Editor. Before an analysis method has been set, the Results Editor
home screen will display the plate layout and thermal cycling program. Each stage where readings have been made
will have its own tab showing a graph of raw data.

Each stage where readings have been made will have a separate results
tab enabling analysis of the data for that particular stage.

The Analysis Selection box displays the stage name as assigned in the
program setup. Only those stages that have been assigned with reads are
displayed since stages without reads have no data to analyse. Double-click
on a stage name, or highlight a stage name then click on Edit to launch the
Analysis Wizard Selection screen.

Selecting the Analysis Method

The Analysis Selection box on the Results Editor home screen allows the user to define the method of analysis to
be applied to the readings gathered during the PCR run. Highlighting a stage name and pressing the Edit button will
launch the Analysis Wizard Selection screen and allow an Analysis Method to be assigned for that stage.

Figure 2: The Analysis Wizard Selection screen.

Analysis Method: The drop-down menu lists analysis types appropriate to

the selected stage. These will be dependent on the number of reads and
the number of cycles programmed into the run.
Dye name: The name of the dyes selected in the program setup will be
displayed.
Dye Usage: Assign a Dye Usage from the list in the drop-down menu. There
will be one dye usage box for each read present in the stage.

Cancel: Aborts the procedure and takes the user back to the Results Editor.
Finish: Accepts all the default analysis settings for the analysis method

chosen and closes the Wizard.

Reset defaults: Returns all analysis parameter settings to the defaults.
Back/Next: Allows the user to move between screens in the Analysis

Wizards.

The Analysis Wizards

Once an analysis method has been defined, a series of default settings will automatically analyse the data. The
defaults can be viewed in the Analysis Wizards and edited if required. The intuitive Analysis Wizards explain in
detail the mathematics behind the analysis settings and will lead you through each stage of the analysis setup.
With experience, analysis can be quickly performed using the Analysis Properties displayed in the Parameters (PAR)
box feature found on each data graph.

PrimeQHelp@bibby

-

scientific.com

|www.bibby

-

scientific.com

[phone]

2

T08-002A: Relative Quantification Analysis

Analysis Method Options

The following analysis options are available in Quansoft:

Baseline

This simple analysis method allows for correction of differences in background fluorescence. It is also incorporated
into many of the other analysis methods.

Quantification

Quantification analysis is used to determine the absolute or relative quantity of a target DNA template in a given
test sample by measuring the cycle-to-cycle change in the fluorescent signal. The fluorescent signal increases
proportionately to the amount of amplified DNA and quantification is performed either by comparison of the
fluorescence of a PCR product of unknown concentration with that of several dilutions of an external standard, or
by comparing the fluorescence of one product relative to another. To be able to make this comparison, the
fluorophore is measured at a point in the amplification where the reaction efficiency can be considered optimal.
This is generally around the cycle at which an increase in fluorescence is first detected.

Dissociation curve

Dissociation curve analysis can add to the information obtained from the PCR. Also known as melting curve
analysis, it measures the temperature at which the DNA strands separate into single strands. This provides a
measurement of the melting temperature or Tm, taken as the point at which 50% of the double stranded DNA
(dsDNA) molecules are dissociated. Using the easy-to-program ‘ramp’ function, PrimeQ will perform a thermal
ramping program that can be used to determine the Tm of the PCR product. This analysis provides the user with
extra confidence in experiments using intercalating dye chemistry for identifying amplification of non-specific
products or contamination.

Plus/minus scoring

This analysis exploits PrimeQ’s fluorescence technology to determine with ease and accuracy the presence or
absence of a PCR product in any given sample. Input data can either be kinetic (where readings are taken
throughout the amplification stage) or end-point (readings taken at the end of the run). The software scores the
samples as positive or negative according to user-defined thresholds.

Allelic discrimination

Users of PrimeQ have the option of this powerful technique capable of detecting single nucleotide differences
(SNPs). It can be used to discriminate between genotypes, mutations and polymorphisms within or between
samples simply by comparing the fluorescence signal obtained using allele-specific, dye-labeled probes.

Multi-read

This is a simple end point analysis method which will report the average fluorescence of a selected number of
readings. It is useful for assays other than PCR, for example fluorescence-based DNA assays, where just the
fluorescence of a sample needs to be measured; in this way PrimeQ can be used as a simple fluorescence plate
reader or fluorimeter.

PrimeQHelp@bibby

-

scientific.com

|www.bibby

-

scientific.com

[phone]

3

T08-002A: Relative Quantification Analysis

Absolute

quantification

Uses known standards to generate a standard curve and calculates the “absolute”

Relative quantification

Uses known standards to generate a standard curve for each reporter and compares

Relative Cq

Subtracts the Cq value

of a reference from

an unknown amplified from the same

Analysis method: Relative Quantification

This analysis method determines the concentration of one target relative to another and is often used to compare
the expression of a gene of interest with that of a reference or housekeeping gene amplified from the same
sample. Automatic relative quantification analysis in Quansoft can only be applied to a multiplex reaction where
the two targets are amplified in the same well. This ensures that the amount of sample added to the well is
consistent for both the gene of interest and reference.

There are two methods available in Quansoft to compare the relative amounts of two samples. Firstly, Relative
Quantification is similar to absolute quantification analysis but uses two reporter dyes and two standard curves to
compare the concentration of one DNA template relative to a second template. This method is useful for
optimizing reactions for multiplex assays. Secondly, Relative Cq is used in the comparison of the relative amount of
samples in different wells. This approach is particularly useful for screening assays where it is necessary to compare
a fold difference of sample B to a calibrator sample A, for example. In such an assay, information about absolute
amounts is not required as the value relative to the calibrator provides the necessary information. No standards
are required. This is commonly known as the 2

Both approaches use quantification cycle (Cq) calculation as the basis of the quantification. The Cq is the cycle
number at which the concentration of the amplicon (measured by fluorescence) reaches a set threshold. The Cq is
inversely proportional to the initial template concentration. Quansoft can use one of two methods to calculate Cq
values: fit points or first derivative maximum; further details are given below.

-ΔΔCq

method.

value of the unknowns e.g. copies/well, µg/ml etc.

the absolute concentrations of the two reporters in the same well (ratio of one to the
other).

sample and compares to a control sample (2

Table 1: Quantification analysis options. Relative quantification methods are described in this Technical Note.

-ΔΔCp

method).

Setting up and running the experiment

Quantification analysis is a kinetic analysis method and requires fluorescence readings to be taken at each cycle of
the amplification stage. Therefore a reading for each reporter and a passive reference dye (PRD), if used must be
included at the end of the extension step of each cycle.

For relative quantification using a standard curve, standards of know concentration or copy number amplified by
the same primers as the target must also be prepared. This is to ensure that the reaction efficiency is the same
between the standards and unknowns. We recommend that you use at least three standards diluted serially at 10­fold dilutions. For increased precision, all reactions should be performed in duplicate or triplicate. The standards
need to be defined in the plate layout and the concentrations added to the Well Information table in the Plate
Layout Editor. To enable the relative quantification analysis, standards of the same concentration for each reaction
of the multiplex must be amplified in the same wells. For example, if standards of 1000, 10,000 and 100,000 copies
for reaction A are placed in wells B1, C1 and D1, then standards of 1000, 10,000 and 100,000 copies for reaction B
must also be amplified in wells B1, C1 and D1. The same applies to unknowns.