Real Time (q)PCR Range

Real-time PCR (also known as quantitative PCR or qPCR) is a powerful and common technique for accurate analysis of gene expression. It is used to amplify and simultaneously quantify a targeted DNA molecule. Unlike traditional PCR, which involves running an electrophoresis gel at the end to visualize the amplified products, real-time PCR enables the detection and quantification of nucleic acids during the PCR process. This allows for the continuous tracking of the reaction's progress and accurate quantification of the nucleic acids as they are being amplified.

Principle of probe-based qPCR

• Oligonucleotides modified with a 5’ fluorophore (e.g., FAM) and a 3’ quencher (e.g., TAMRA) are added to the reaction. Under annealing conditions, the probe hybridizes in a sequence-specific manner to the template DNA. Fluorescence of the fluorophore is suppressed by the quencher. During the extension reaction, the 5’→ 3’ exonuclease activity of Taq DNA polymerase degrades the hybridized probe, releasing quencher suppression and allowing fluorescence (Figure 1). To maximize sensitivity, our kits use TaKaRa Ex Taq DNA Polymerase Hot-Start Version, a hot-start PCR enzyme that minimizes nonspecific amplification that may arise from mispriming or primer-dimer formation during reaction mixture preparation and pre-cycling steps.

• The use of highly specific probes ensures accurate detection of target sequences with minimal interference.

• Multiplexing Capability: Unique labeling of each probe allows for the simultaneous amplification of multiple targets in the same reaction tube, increasing throughput.

• Multiplexing minimizes the need for multiple reactions, reducing the handling and preparation time of samples.

• The probes can precisely discriminate between different single nucleotide polymorphisms (SNPs) and copy number variations (CNVs).

• Developing and optimizing probes can be time-consuming and may require several iterations to ensure specificity.

• Probes, which include both a fluorophore and a quencher, are more expensive than simple oligonucleotide primers. Pre-designed probes can have even higher costs.

Fluorescent detection using intercalating dyes

• This method uses a DNA intercalator (e.g., TB Green) that emits fluorescence when bound to double-stranded DNA. Monitoring fluorescence allows for quantification of amplification products (Figure 1). Following amplification, performing a melt curve analysis provides information on the specificity of your PCR products. To maximize specificity and sensitivity, our kits use Takara Ex Taq DNA Polymerase Hot-Start Version, a hot-start PCR enzyme that minimizes nonspecific amplification that may arise from mispriming or primer-dimer formation during reaction mixture preparation and pre-cycling steps.

• It uses standard, unlabeled oligonucleotides, making it less expensive than probe-based methods.

• The method is straightforward and requires less optimization compared to probe-based qPCR.

• It is well-suited for high-throughput screens and large-scale studies due to its ease of use and lower cost.

• Despite being cost-effective, dye-based qPCR can still provide sensitive detection for gene expression studies.

• The intercalating dye binds to all double-stranded DNA, including nonspecific PCR products, which can lead to false positives.

• To assess the specificity of the amplification, a melt curve analysis is often required, adding extra hands-on time and potential complexity.

• Unlike probes, predesigned dye-based primer sets are less commonly available, requiring the user to design and validate their own primers.

When starting with RNA samples, one must first perform a reverse transcription (RT) step to generate cDNA for the subsequent qPCR reaction. One-step RT-qPCR streamlines this workflow by performing the RT step in the same tube as the qPCR reaction 

• Simple and rapid workflow

• Compatible with large numbers of samples (when looking at few target genes)

• Adaptable to high-throughput/automated workflows

• Cannot optimize RT step

• Does not generate stock cDNA

• Not ideal when analyzing large numbers of target genes

Quantitative PCR (qPCR) is a common, powerful technique for the accurate analysis of gene expression. When starting with RNA samples, you must first perform a reverse transcription (RT) step to generate cDNA for the subsequent qPCR reaction. Two-step RT-qPCR performs the RT step in one tube and the qPCR reaction in a separate tube.

• Compatible with limited sample input due to high sensitivity

• Able to optimize RT and qPCR steps separately

• Can generate cDNA stocks

• More time-consuming

• Less amenable to high-throughput workflows

• Uracil-DNA glycosylases (UDGs) are highly conserved enzymes involved in DNA repair across evolution. The UDG family consists of six subfamilies, with Family I UDG enzymes known as UNG, named after the uracil-N-glycosylase gene. UDG and UNG are often used interchangeably, as they both serve the same function in qPCR by preventing carryover contamination.

• The primary biological role of UDG is to eliminate uracil, which is typically found in RNA, from DNA, generating free uracil and alkali-sensitive apyrimidinic sites in the DNA. UNG facilitates the removal of uracil from both single- and double-stranded DNA by catalyzing the hydrolysis of the N-glycosidic bond between uracil and the sugar backbone. Notably, the enzyme has a strong preference for acting on single-stranded uracil-containing templates

• Preventing contamination is crucial in qPCR, as even trace amounts of DNA can lead to false positives. Contamination sources include cross-sample transfer, environmental DNA, and carryover from previous amplifications, with the latter being a common cause of false positives. To address this, UNG can degrade amplification products from prior PCR runs, while leaving the original template intact.

• UNG targets single- and double-stranded dU-containing DNA, but dUTP is not affected by it. Taq polymerase and other PCR components remain unaffected by UNG treatment, which only removes carryover products. The dU-containing PCR product behaves like native dT-containing DNA in blotting, cloning, and sequencing, and UNG does not impact most post-PCR analyses, including electrophoretic mobility and ethidium bromide staining. To prevent carryover contamination in qPCR, use a master mix containing UNG or UDG.