Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected throughout the PCR process, rather than at the end of the PCR. This completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA. In real-time PCR (qPCR), reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. In contrast, an endpoint assay (also called a “plate read assay”) measures the amount of accumulated PCR product at the end of the PCR cycle.
We have developed two types of chemistries used to detect PCR products using Sequence Detection Systems (SDS) instruments:
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles.
Assay Types that Use TaqMan Chemistry
The TaqMan chemistry can be used for the following assay types:
SYBR Green I Dye Chemistry
The SYBR Green I dye chemistry uses Applied Biosystems™ SYBR™ Green I dye, a highly specific, double-stranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles. The most important difference between the TaqMan and SYBR Green I dye chemistries is that the SYBR Green I dye chemistry will detect all double-stranded DNA, including non-specific reaction products. A well-optimized reaction is therefore essential for accurate results.
Assay Types that Use SYBR Green I Dye Chemistry
The SYBR Green I dye chemistry can be used for the following assay types:
Initially, intercalator dyes were used to measure real-time PCR (qPCR) products. The primary disadvantage to these dyes is that they detect accumulation of both specific and nonspecific PCR products.
Development of TaqMan Chemistry
Real-time systems for PCR were improved by the introduction of fluorogenic-labeled probes that use the 5´ nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products. The development of fluorogenic labeled probes also made it possible to eliminate post-PCR processing for the analysis of probe degradation
How TaqMan Sequence Detection Chemistry Works
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR. Here’s how it works:
Two Types of TaqMan Probes
We offer two types of Applied Biosystems™ TaqMan® probes:
TaqMan MGB Probes Recommended for Allelic Discrimination Assays
We recommend the general use of TaqMan MGB probes for allelic discrimination assays, especially when conventional TaqMan probes exceed 30 nucleotides. The TaqMan MGB probes contain:
Consequently, the TaqMan MGB probes exhibit greater differences in Tm values between matched and mismatched probes, which provide more accurate allelic discrimination.
Advantages of TaqMan Chemistry
The advantages of the TaqMan chemistry are as follows:
Disadvantage of TaqMan Chemistry
The primary disadvantage of the TaqMan chemistry is that the synthesis of different probes is required for different sequences.
Small molecules that bind to double-stranded DNA can be divided into two classes:
Regardless of the binding method, there are two requirements for a DNA binding dye for real-time detection of PCR:
We have developed conditions that permit the use of the SYBR Green I dye in PCR without PCR inhibition and increased sensitivity of detection compared to ethidium bromide.
How the SYBR Green I Dye Chemistry Works
The SYBR Green I dye chemistry uses the SYBR Green I dye to detect polymerase chain reaction (PCR) products by binding to double-stranded DNA formed during PCR. Here’s how it works:
When SYBR Green I dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.
Advantages of SYBR Green I Dye
The advantages of the SYBR Green I dye chemistry are as follows:
Disadvantage of SYBR Green I Dye
The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green I dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.
Another aspect of using DNA binding dyes is that multiple dyes bind to a single amplified molecule. This increases the sensitivity for detecting amplification products. A consequence of multiple dye binding is that the amount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than a shorter one. This is in contrast to the use of a fluorogenic probe, in which a single fluorophore is released from quenching for each amplified molecule synthesized, regardless of its length.
What Is a Quantitation Assay?
A Quantitation Assay is a real-time PCR (qPCR) 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. There are three types of Quantitation Assays discussed in this chemistry guide:
Terms Used in Quantitation Analysis
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
Ct (threshold cycle): The fractional cycle number at which the fluorescence passes the fixed threshold 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. A passive reference dye is included in all SDS PCR reagent kits.
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:
ΔRn (delta Rn): The magnitude of the signal generated by the given set of PCR conditions.
The ΔRn 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.
How Real-Time PCR (qPCR) 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.
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.
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.
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).
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.
Calculation Methods for Relative Quantitation
Relative quantitation can be performed with data from all of the SDS instruments. The calculation methods used for relative quantitation are:
Determining Which Method to UseAll methods can give equivalent results. When determining which method you want to use, note the following:
The following terms are used in this discussion of absolute and relative quantitation:
Standard: A sample of known concentration used to construct a standard curve.
Reference: A passive or active signal used to normalize experimental results. Endogenous and exogenous controls are examples of active references. 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 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.
Calibrator: A sample used as the basis for comparative results.
It is easy to prepare standard curves for relative quantitation because quantity is expressed relative to some basis sample, such as the calibrator. For all experimental samples, target quantity is determined from the standard curve and divided by the target quantity of the calibrator. Thus, the calibrator becomes the 1 X sample, and all other quantities are expressed as an n-fold difference relative to the calibrator. As an example, in a study of drug effects on expression, the untreated control would be an appropriate calibrator.
The guidelines below are critical for proper use of the standard curve method for relative quantitation:
Amplification of an endogenous control may be performed to standardize the amount of sample RNA or DNA added to a reaction. For the quantitation of gene expression, researchers have used ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA (rRNA), or other RNAs as an endogenous control.
Because the sample quantity is divided by the calibrator quantity, the unit from the standard curve drops out. Thus, all that is required of the standards is that their relative dilutions be known. For relative quantitation, this means any stock RNA or DNA containing the appropriate target can be used to prepare standards.
The comparative CT method is simlar to that standard curve method, except it uses the arithmetic formula, 2−ΔΔCT to achieve the same result for relative quantitation.
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.
For more information on using the comparative CT method for relative quantitation, please refer to User Bulletin #2: Relative Quantitation of Gene Expression (PN 4303859).
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.
The guidelines below are critical for proper use of the standard curve method for absolute quantitation:
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.
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 A260 and converted to the number of copies using the molecular weight of the DNA or RNA.
The PCR process and 5’ nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.
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For Research Use Only. Not for use in diagnostic procedures.