What Is a Quantitation Assay?
A Quantitation Assay is a real-time PCR assay. It measures (quantitates) the amount of a nucleic acid target during each amplification cycle of the PCR. The target may be DNA, cDNA, or RNA.
How Real-Time PCR Quantitation Assays Work
In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence above the baseline indicates the detection of accumulated target. A fixed fluorescence threshold can be set above the baseline. The parameter CT (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.
Overview
When calculating the results of your quantitation assays, you can use either absolute or relative quantitation.
What is Absolute Quantitation?
The absolute quantitation assay is used to quantitate unknown samples by interpolating their quantity from a standard curve.
Example
Absolute quantitation might be used to correlate viral copy number with a disease state. It is of interest to the researcher to know the exact copy number of the target RNA in a given biological sample in order to monitor the progress of the disease.
Absolute quantitation can be performed with data from all of the SDS instruments, however, the absolute quantities of the standards must first be known by some independent means.
Standard Curve Method for Absolute Quantitation
Overview
The standard curve method for absolute quantitation is similar to the standard curve method for relative quantitation, except the absolute quantities of the standards must first be known by some independent means.
Critical Guidelines
The guidelines below are critical for proper use of the standard curve method for absolute quantitation:
- It is important that the DNA or RNA be a single, pure species. For example, plasmid DNA prepared from E. coli often is contaminated with RNA, which increases the A260 measurement and inflates the copy number determined for the plasmid.
- Accurate pipetting is required because the standards must be diluted over several orders of magnitude. Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate A260 value. This concentrated DNA or RNA must then be diluted
- 106–1012 -fold to be at a concentration similar to the target in biological samples.
- The stability of the diluted standards must be considered, especially for RNA. Divide diluted standards into small aliquots, store at –80 °C, and thaw only once before use.
- It is generally not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription step.
Standards
The absolute quantities of the standards must first be known by some independent means. Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute standards. Concentration is measured by A 260 and converted to the number of copies using the molecular weight of the DNA or RNA.
What is Relative Quantitation?
A relative quantitation assay is used to analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
Example
Relative quantitation might be used to measure gene expression in response to a chemical (drug). The level of gene expression of a particular gene of interest in a chemically treated sample would be compared relative to the level of gene expression an untreated sample.
Comparative CT method for Relative Quantitation
The comparative CT method is similar to that standard curve method, except it uses the arithmetic formula, 2- DD CT to achieve the same result for relative quantitation.
Arithmetic Formulas:
For the comparative CT method to be valid, the efficiency of the target amplification (your gene of interest) and the efficiency of the reference amplification (your endogenous control) must be approximately equal.
Terms Used in Quantitation Analysis
Active reference - Active reference means the signal is generated as the result of PCR amplification. The active reference has its own set of primers and probe.
Endogenous and exogenous controls are examples of active references.
Amplicon A short segment of DNA generated by the PCR process.
Amplification plot The plot of fluorescence signal versus cycle number.
Baseline The initial cycles of PCR, in which there is little change in fluorescence signal.
Calibrator A sample used as the basis for comparative results.
CT (threshold cycle) The fractional cycle number at which the fluorescence passes the fixed threshold
Endogenous control – This is an RNA or DNA that is present in each experimental sample as isolated. By using an endogenous control as an active reference, you can normalize quantitation of a messenger RNA (mRNA) target for differences in the amount of total RNA added to each reaction.
Exogenous control – This is a characterized RNA or DNA spiked into each sample at a known concentration. An exogenous active reference is usually an in vitro construct that can be used as an internal positive control (IPC) to distinguish true target negatives from PCR inhibition. An exogenous reference can also be used to normalize for differences in efficiency of sample extraction or complementary DNA (cDNA) synthesis by reverse transcriptase. Whether or not an active reference is used, it is important to use a passive reference containing the dye ROX in order to normalize for non-PCR-related fluctuations in fluorescence signal.
Normalized amount of target - A unitless number that can be used to compare the relative amount of target in different samples.
NTC (no template control) - A sample that does not contain template. It is used to verify amplification quality.
Nucleic acid target (also called “target template”) - DNA or RNA sequence that you wish to amplify
Passive reference - A dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for forestallment fluctuations caused by changes in concentration or volume.
Reference A passive or active signal used to normalize experimental results.
Rn (normalized reporter) - The fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye
Rn+ The Rn value of a reaction containing all components, including the template
Rn- The Rn value of an un-reacted sample. The Rn- value can be obtained from:
- The early cycles of a real-time PCR run (those cycles prior to a detectable increase in fluorescence), OR
- A reaction that does not contain any template
ΔRn (delta Rn) - The magnitude of the signal generated by the given set of PCR conditions. The Δ Rn value is determined by the following formula: (Rn+) – (Rn-)
Standard A sample of known concentration used to construct a standard curve. By running standards of varying concentrations, you create a standard curve from which you can extrapolate the quantity of an unknown sample.
Threshold The average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor. The threshold should be set in the region associated with an exponential growth of PCR product.
Unknown A sample containing an unknown quantity of template. This is the sample whose quantity you want to determine.