The Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) is a highly sensitive method used to measure the amount of a specific RNA sequence, typically messenger RNA (mRNA), within a biological sample. This technique quantifies gene expression levels, providing insights into how genes are regulated. RT-qPCR is a two-step process: first, RNA is converted into complementary DNA (cDNA) via reverse transcription, followed by the exponential amplification and quantification of the cDNA using quantitative Polymerase Chain Reaction (qPCR).
RNA Isolation and Quality Assessment
High-quality and intact RNA is necessary for successful RT-qPCR, as degraded or contaminated RNA leads to inaccurate quantification. RNA isolation typically uses column-based kits or extraction reagents like Trizol, which denature proteins and inactivate RNases. Proper handling, including the use of RNase-free consumables, is required because RNA is highly susceptible to degradation.
After isolation, a spectrophotometer measures the RNA concentration and purity. The A\(_{260}\)/A\(_{280}\) ratio assesses protein contamination; pure RNA samples exhibit a ratio between 1.8 and 2.1. The A\(_{260}\)/A\(_{230}\) ratio detects contamination from organic solvents or salts, with values generally expected to be greater than 1.7.
RNA integrity is measured using the RNA Integrity Number (RIN), generated by automated electrophoresis systems, where a value of 10 indicates completely intact RNA. RIN values of 8.0 or above are preferred for reliable results. A DNase treatment step is also performed to eliminate contaminating genomic DNA (gDNA), preventing false positive results during the qPCR step.
The Reverse Transcription Step
Reverse transcription converts the single-stranded RNA template into a stable complementary DNA (cDNA) molecule, which serves as the template for qPCR. This process is catalyzed by reverse transcriptase, which synthesizes the first cDNA strand. The choice of primer used to initiate synthesis significantly impacts the final cDNA pool.
Primer Types
Oligo(dT) primers anneal specifically to the poly-A tail of eukaryotic mRNA, ideal for focusing on mRNA. However, this can cause a 3′ bias, resulting in shorter cDNA transcripts. Random hexamer primers bind non-specifically across the entire RNA population, yielding a more representative, fragmented cDNA pool. These are often used for degraded or non-polyadenylated RNA. Gene-specific primers (GSPs) target only the RNA sequence of interest, offering the highest specificity for low-abundance targets. Some commercial kits use a mixture of oligo(dT) and random hexamers for comprehensive coverage. Reaction conditions, including temperature, are optimized to minimize secondary RNA structures that impede synthesis.
Setting Up the Amplification Reaction
The qPCR phase involves the exponential amplification of the cDNA template and simultaneous monitoring of product accumulation using a thermal cycler with a fluorometer. The reaction mixture requires several components: the cDNA template, forward and reverse primers, dNTPs, a heat-stable DNA polymerase, a reaction buffer, and a fluorescent reporter system. Reporters are typically either a non-specific DNA-binding dye (e.g., SYBR Green) or a sequence-specific fluorescent probe.
Primer Design
Primer design balances specificity with amplification efficiency. Primers should be short (18 to 30 bases) and have similar melting temperatures (T\(_{m}\)), typically 60–65°C, for efficient annealing. Primers must be specific to the target sequence and designed to avoid forming secondary structures or primer-dimers, which interfere with quantification. A common technique to prevent residual genomic DNA amplification is designing primers to span an exon-intron boundary.
Thermal Cycling
The thermal cycling protocol consists of three main steps repeated for 35 to 40 cycles. Denaturation occurs at high temperature (around 95°C) to separate the double-stranded DNA template. Next, the annealing step lowers the temperature (usually 5°C below the primer T\(_{m}\)) allowing primers to bind. Finally, the extension step (typically 72°C) allows the DNA polymerase to synthesize a new DNA strand.
Essential Controls
The experiment must include controls to validate results. A No Template Control (NTC) checks for contamination in reagents or plastics by including all components except the cDNA. A No Reverse Transcriptase Control (No-RT Control) verifies that DNase treatment successfully removed genomic DNA; amplification here indicates gDNA contamination. Additionally, a reference gene (housekeeping gene) is amplified alongside the target gene to standardize the amount of starting material for relative quantification.
Interpreting Data and Validating Results
The primary output of a qPCR run is amplification curves plotting the increase in fluorescent signal against the number of thermal cycles. The Cycle threshold (Ct), or quantification cycle (Cq), is the cycle number where the fluorescent signal crosses a set threshold. The Ct value is inversely related to the initial amount of target RNA; a lower Ct indicates a higher starting concentration.
Gene expression quantification uses two methods. Absolute quantification determines the exact copy number by comparing sample Ct values to a standard curve of known concentrations. Relative quantification, common in gene expression studies, measures the change in expression relative to a control condition. This method uses the Ct values of the target gene and a stable reference gene to calculate the fold change.
For reactions using non-specific dyes like SYBR Green, a melt curve analysis confirms product specificity. This analysis involves gradually raising the temperature and monitoring fluorescence decrease as the double-stranded DNA melts; a single, sharp peak indicates a specific product. Reaction efficiency is also evaluated, ideally falling between 90% and 110%, confirming that the product approximately doubles with each cycle.