qPCR
Glossary
Absolute Quantification: Calculate the exact number of copies of the target sequence using a standard curve.
Amplification Efficiency Correction: Normalizing quantification figures according to the amplification efficiency of the qPCR reaction.
Amplification Efficiency Plot: A plot depicting the correlation between the cycle number and the log of template concentration, to evaluate the qPCR reaction efficiency.
Amplification Plot: A plot reporting the intensity of the fluorescent signal (from the Probe or intercalating dye) during each PCR cycle, allowing the user to follow the reaction in real time.
Annealing: Annealing is an important step in PCR enabling the specific binding of the Oligonucleotides (primers) to their complementary target sequence at a specific temperature. The presence of annealed primers is essential for initiating the elongation phase of PCR.
Baseline and Threshold: The baseline is the average background, and the threshold is chosen value of fluorescence above the baseline, at which the signal can be considered not to be background.
cDNA: cDNA, meaning "complementary DNA", is a synthesized DNA strand from a complementary messenger RNA template.
Core Kit: A Core kit, is a kit that contains all components in separate tubes, so you have to mix them yourself. It gives a maximum of flexibility, as you can optimize the concentration of each component of your assay.
Cq/Ct: Cq/Ct, or quantification cycle/threshold, is the PCR cycle where the sample amplification curve intersects the threshold. This is therefore the number of PCR cycles necessary for the sample to be identifiable and/or measurable out of the background.
Data Normalization Methods: Diverse methods for standardizing qPCR data include utilizing reference genes, total RNA input, or incorporating spike-in controls.
Delta Rn (ΔRn): The difference between the fluorescence signal of the reporter dye and the baseline fluorescence.
Denaturation: This is the first step of PCR. In this step, heat is applied to the template to reach 94-98°C. At this temperature, the double-stranded DNA separate into two single strands. Following the denaturation step is the annealing step.
Double-dye Probe: Double-Dye Oligonucleotides have a fluorescent reporter dye and a quencher at their 5’ and 3’ ends, respectively.
Dynamic Range: The range of template concentrations within which qPCR can precisely quantify nucleic acids.
Efficiency Calculation: The efficiency of the PCR reaction, expressed as a percentage, indicating how much the amount of target doubles with each cycle. It's calculated from the amplification plot.
Enzyme activation: Activation time refers to the length of time that elapses before the enzyme is activated at 95'C.
Fluorophore: Molecule that can absorb light energy through excitation and subsequently release light energy at a longer wavelength, a phenomenon known as emission.
FRET: Förster Resonance Energy Transfer is a widely used technology. It relies on the energy transfer between 2 molecules (2 fluorophores or one fluorophore and a quencher). In close proximity the fluorophore 2 (or Quencher) can absorb the energy emmitted by the excited fluorophore 1 and start to fluoresce.
HotStart: Hot start PCR is a variant of the polymerase chain reaction (PCR) developed to suppress premature enzymatic activity. In this method there is a controlled chemical or physical activation of the enzyme. This technique avoids non-specific amplification of DNA.
Master Mix: a MasterMix is a ready-to-use reagent in which all the components are already mixed in an optimized way. Additional stabilizers are added to enable long-term storage.
Melting Curve Analysis: Melting curve analysis allows to discriminate between specific and non-specific amplification products. This is a technique based on the DNA dissociation profile by slow increase of the temperature.
MGB Probe: Minor Groove binding probes are a type of qPCR Probes with an additional MGB moiety. The MGB moiety binds to the minor groove of the target DNA. It offers a higher specifcity and stability to the probe.
MIQE guidelines, or MIQE: MIQE constitutes a set of recommendations outlining the essential information required to assess qPCR experiments, and submit article for publication.
Multiplex qPCR: Conducting simultaneously several qPCR reactions to identify and measure multiple target sequences within a single tube of sample.
Nonspecific Amplification: Amplification products that are not the intented target, frequently resulting from primer-dimer formation or non-specific binding.
Non Template controls: A sample or reaction that does not contain the target nucleic acid sequence intended to amplify. It is used as a reference point to ensure the specificity and accuracy of the PCR reaction.
Normalization: Adjustment of qPCR data to accommodate variations. It is typically achieved through the utilization of specific dyes (e.g. ROX™), reference genes or internal controls.
One-step RT-qPCR: This method refers to a reverse transcription quantitative polymerase chain reaction where both the reverse transcription of RNA into complementary DNA and the subsequent PCR amplification occur within a single reaction tube.
Plateau Phase: The attenuation in the rate of the exponential product accumulation, indicating that most of the target has been amplified.
Polymerase: An enzyme responsible for producing extended chains of nucleic acids.
Polymerase Chain Reaction (PCR): The principle, and aim, of the PCR technology is to specifically increase undetectable amount of DNA starting material, creating sufficient material for detection and analysis.
Positive controls: A sample or reaction that contains a known quantity of the target nucleic acid sequence you intend to amplify. It serves as a reference to ensure that the PCR reaction is functioning correctly and that the amplification process is producing the expected results.
Primer Dimers and Non-specific Amplification: Two issues that can arise during PCR and qPCR due to primer self-annealing or non-specific binding of primers to unintended sequences.
Primer: Short DNA fragments that serves as a starting point for a PCR reaction. Primers are a crucial component of PCR because they define the sequence to be amplified.
Probe: A single-stranded DNA or RNA molecule labeled with dyes (most of the time a fluorophore and a quencher) and designed to specifically bind to a complementary sequence of DNA or RNA in order to visualize or measure the presence of the target sequence.
qPCR baseline: Refers to the initial phase of the fluorescence curve recorded during the amplification process.
Quantitation Cycle (Cq): A term sometimes used interchangeably with Ct, representing the cycle at which the amplification curve crosses a specific threshold.
Quantitative PCR (qPCR): A method employed to measure the quantity of DNA or RNA in a sample. This involves amplifying DNA fragments and monitoring their fluorescence during the amplification process.
Quencher: Quencher is a molecule that can absorb and disipate energy emitted by a fluorophore and reduce the fluorescent intensity.
Real-time Monitoring: Tracking fluorescence in every amplification cycle, enabling real-time observation of the advancement of the amplification reaction.
Relative Quantification: Assessing the level of target gene expression across distinct samples, commonly standardized against a reference gene or control sample.
Replicates: Performing qPCR reactions repeatedly for every sample to accommodate experimental fluctuations and enhance the dependability of outcomes.
Reproducibility: Reproducibility refers to the ability of an experiment or study to yield consistent and comparable results when repeated by different researchers, using different instruments or equipment, or conducted at different times or locations.
Reverse transcriptase: Reverse transcriptase is an enzyme employed to create complementary DNA from an RNA template, a procedure known as reverse transcription.
Reverse Transcription (RT): The conversion of RNA into complementary DNA (cDNA) by the enzyme reverse transcriptase. This stage is essential when analyzing RNA targets by qPCR.
ROX (Reference Dye): ROX is a member of the Rhodamine family and is frequently used as a reporter dye, especially for fluorescent DNA sequencing and RT-qPCR, to standardize fluorescence variations and variations in instrument performance.
RT-qPCR: Reverse transcription-qPCR is a real-time PCR technique using RNA as the initial template. The RNA is first reverse transcribed into cDNA before the PCR amplification.
Sensitivity: It reflects the method's capacity to identify even subtle variations in the quantity or presence of the target being measured.
Singleplex: Used to detect a single target of DNA or RNA.
SNP (pronounced snip): Single-nucleotide polymorphism (SNP) is a type of genetic variation occuring at a single base position. It can impact an individual's vulnerability to diseases, reactions to medications, or even be employed to track the inheritance of particular genes and traits of significance.
Standard Curve: A graph showing the correlation between cycle threshold (Ct) values of established DNA/cDNA concentrations and the logarithm of these concentrations.
SYBR® Green: SYBR® Green is an inexpensive, universal dye which binds to dsDNA.
Thermocyclers: Devices to amplify DNA samples through the polymerase chain reaction.
Threshold Cycle (Ct): An alternative expression for the cycle threshold, denoting the cycle when the fluorescence signal intersects the threshold level.
Two-step RT-qPCR: RT-qPCR that carries out in 2 steps. The first one is the reverse transcription of RNA to cDNA. The second one uses a part of the cDNA as the template for subsequent PCR amplification.