Techne PrimeQ Relative Quantification Analysis

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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.
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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.
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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.
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