Quantitative Polymerase Chain Reaction (qPCR) measures the amount of a specific DNA sequence in a sample. Unlike traditional PCR, qPCR monitors DNA amplification in real-time. This is achieved using fluorescent dyes or probes that emit light as DNA synthesizes. SYBR Green I is a common fluorescent dye that simplifies detection. It binds to any double-stranded DNA, and as more DNA is produced, fluorescence intensity increases proportionally, providing a measurable signal.
Essential Components and Reagents
SYBR Green Master Mix
The SYBR Green Master Mix is a pre-formulated solution containing most necessary reagents. It includes Taq polymerase, a heat-stable enzyme that synthesizes new DNA strands by extending primers. Deoxynucleotide triphosphates (dNTPs), the building blocks of DNA, are also part of the mix. A reaction buffer maintains optimal pH and ionic conditions for enzyme activity, and the SYBR Green I dye allows for fluorescence detection. Many master mixes also contain a passive reference dye, like ROX, to normalize well-to-well optical variations.
Primers
Primers are short, synthetic DNA sequences, 18 to 25 nucleotides long, designed to bind specifically to the target DNA sequence. Both a forward and a reverse primer are required to define the region for amplification. Good primer design is important for accurate results. Primers should have a melting temperature (Tm) between 58°C and 60°C, with minimal difference between forward and reverse primers for synchronized binding during annealing. Primers are also designed to avoid internal secondary structures, such as hairpins or self-dimers, and to have a balanced GC content (40-60%) to promote stable binding.
Template Nucleic Acid
The starting material for qPCR is the template nucleic acid, either DNA (genomic DNA) or complementary DNA (cDNA) derived from RNA. The template’s purity and concentration are important for reliable quantitative data. Contaminants like proteins or residual chemicals can inhibit the PCR reaction, leading to inaccurate results. Optimal template concentrations range from 1–10 ng of cDNA or 10–100 ng of genomic DNA per reaction.
Nuclease-Free Water
Nuclease-free water adjusts reaction volume and dilutes reagents. It is also used in “no template control” (NTC) reactions, serving as a negative control to detect contamination. An NTC reaction should show no amplification, confirming the absence of contaminating DNA.
Reaction Assembly and Thermal Cycling
Reaction Assembly
All reagents, including the SYBR Green Master Mix, primers, template nucleic acid, and nuclease-free water, should be thawed on ice and briefly centrifuged. Preparing a master mix containing all common reagents (master mix, primers, and water) for all samples, excluding the template, is recommended. This minimizes pipetting variability and ensures consistency.
After preparing the master mix, aliquot it into individual PCR tubes or wells. Then, add the specific template DNA or cDNA to each sample, and nuclease-free water to no-template control wells. Gentle mixing, followed by brief centrifugation, ensures components are combined. Maintaining a clean workspace and using sterile, filter-tipped pipettes prevents contamination and false positives.
Thermal Cycling Program
Once assembled, reactions are placed into a real-time PCR instrument, or thermocycler, which controls temperature changes for DNA amplification and fluorescence detection. The program begins with an initial denaturation step at 95°C for 2-5 minutes. This activates Taq polymerase and separates double-stranded DNA templates into single strands for primer binding.
Following initial denaturation, the cycling phase commences for 40 cycles. Each cycle consists of two main steps: denaturation and annealing/extension. Denaturation occurs at 95°C for 5-15 seconds to separate newly formed double-stranded DNA.
The subsequent annealing/extension step combines two processes at 60°C for 30-60 seconds. During this phase, primers bind to separated DNA strands, and Taq polymerase synthesizes new DNA. Fluorescence is measured at the end of this step in each cycle, as SYBR Green binds to newly synthesized double-stranded DNA, allowing real-time monitoring.
Melt Curve Analysis for Specificity
Following amplification, a melt curve analysis confirms PCR product specificity. This is relevant for SYBR Green qPCR because the dye binds to any double-stranded DNA, not just the target amplicon. Non-specific products, like primer-dimers (short DNA fragments formed by primers annealing to each other), would also generate a fluorescent signal, potentially skewing results.
The analysis involves slowly increasing the temperature of amplified samples from 60°C to 95°C, while continuously monitoring fluorescence. As temperature rises, double-stranded DNA products denature, or “melt,” into single strands. When DNA denatures, SYBR Green is released, decreasing fluorescence. The instrument plots the rate of change in fluorescence against temperature, resulting in a melt curve with distinct peaks.
A single, sharp peak indicates only one specific PCR product was amplified, which is the desired outcome. The temperature of this peak, the melting temperature (Tm), is characteristic of the specific amplicon due to its unique sequence and length, falling between 80°C and 90°C for qPCR products. Multiple peaks suggest more than one double-stranded DNA product, such as non-specific amplification or primer-dimers. A peak at a lower temperature (60°C-75°C) indicates primer-dimers, while a peak above the expected amplicon Tm suggests genomic DNA contamination. Identifying these peaks is important for troubleshooting and ensuring only the intended target is quantified.
Interpreting qPCR Data
The Amplification Plot
The amplification plot displays fluorescence intensity against cycle number. This plot exhibits an S-shaped curve for each sample, reflecting PCR reaction progression. Initially, during the baseline phase (cycles 0-10), fluorescence remains low, representing background signal. As amplification proceeds, the fluorescence signal rises exponentially, indicating target DNA doubling in each cycle. This is followed by a linear phase where amplification efficiency decreases, leading to a plateau phase where reaction components are depleted or enzyme activity is limited, and fluorescence no longer increases significantly.
The Quantification Cycle (Cq/Ct Value)
The Quantification Cycle (Cq or Ct) is a numerical value derived from the amplification plot. It represents the cycle number at which a sample’s fluorescence signal crosses a predetermined threshold line, set above background noise in the exponential phase. A lower Cq value directly correlates with a higher amount of starting template nucleic acid. Conversely, a higher Cq value indicates a lower initial quantity. For instance, a difference of approximately 3.32 cycles in Cq value between two samples suggests a tenfold difference in their initial template quantities, assuming 100% amplification efficiency.
Quantification
Quantification in qPCR involves comparing a gene of interest’s expression levels relative to a stable reference gene, also known as a housekeeping gene. Reference genes are chosen because their expression levels are consistent across different samples or experimental conditions. They serve as an internal control to normalize for variations in RNA input or reverse transcription efficiency.
A common approach for calculating relative changes in gene expression is the delta-delta Cq (ΔΔCq) method. This method compares the Cq values of the gene of interest to those of the reference gene within each sample (delta Cq). It then compares these normalized values between a treated or experimental sample and a control sample (delta-delta Cq). The final result is expressed as a fold change, indicating how much the gene’s expression has increased or decreased relative to the control.