Polymerase Chain Reaction (PCR) was developed by Dr. Kary Mullis in the 80s of the 20th century. The technology has been likened to a "molecular copier" for its ability to recognize specific DNA sequences and synthesize large numbers of copies quickly and accurately. Various derivatization techniques based on the original PCR method have been developed. Real-time PCR, also known as quantitative PCR (qPCR), combines PCR amplification and detection in a single step. Another technique called reverse transcription polymerase chain reaction (RT-PCR) uses RNA as a nucleic acid starting template.

● PCR: Repeated heating and cooling cycles of a reaction mixture containing DNA template, DNA polymerase, primers, and dNTPs. [1] Each cycle doubles or so the amount of DNA, as a new strand of DNA becomes a template for replication in the next cycle. This results in an exponential increase in the amount of DNA.

 

Fig.1 First cycle of PCR[1]

 

● RT-PCR (Reverse transcription PCR): Reverse transcription PCR can use RNA as a template to generate complementary DNA (cDNA). Reverse transcriptase is used to generate single-stranded copies of cDNA. This can be done using oligomeric (dT) primers that bind to the PolyA tail of the RNA, or using random hexamers (primers six to nine bases long that bind at multiple points in the RNA transcript). In general, a mix of the two primers is considered best because of its ability to amplify PolyA-tailed RNAs (mostly mRNAs) and PolyA-free RNAs (tRNA, rRNA, etc.). DNA polymerase is then amplified to generate double-stranded cDNA that is fed into a standard PCR-based amplification process.

● qPCR (Quantitative PCR): refers to quantitative real-time PCR, that is, PCR amplification of DNA in real time, and measurement by fluorescent probes (most commonly intercalation dyes or hydrolysis probes) to quantify PCR products. It is used to detect the presence of pathogens and to determine the copy number of the DNA sequence of interest. SYBR Green I is the most commonly used DNA-binding fluorescent dye that fluoresces when bound to double-stranded DNA, and the fluorescence intensity increases in proportion to the concentration of the PCR product.

The advantage of quantitative PCR over standard PCR is that it is possible to observe in real time which reactions are working without the need for an agarose gel. It also enables true quantification.

● RT-qPCR (Reverse Transcription Quantitative real-time PCR): This is a technique that combines RT-PCR with qPCR for reverse transcription quantitative real-time PCR. Rapid detection of changes in gene expression by using cDNA in qPCR reactions to measure RNA levels.

RT-qPCR can be used to study changes in gene expression when a model system is treated with inhibitors, stimulators, small interfering RNAs (siRNAs), or knockout models. This technique is also frequently used to detect expression changes before (as quality control) and after (confirmation of changes) in RNA-Seq experiments.

The final step in RT-qPCR sample preparation is to generate cDNA. In addition to considering primers, cDNA generation can be generated as part of a qPCR experiment (referred to as one-step RT-qPCR) or separately from qPCR (two-step RT-qPCR) (Figure 3).

 

method	One-step RT-qPCR	Two-step RT-qPCR
merit	Experiments have less variability, less risk of contamination, and enable high-throughput screening	More reactions and flexible priming options for each sample
shortcoming	The number of sample uses is limited	More optimization is needed
apply	Clinical screening	Large-scale gene expression analysis

 

 

Fig.2. Schematic diagram of RT-PCR, qPCR and RT-qPCR [2]

 

Fig.3. One-step RT-qPC and two-step RT-qPCR[2]

 

● ddPCR (Droplet-based Digital PCR): The sample passes through a microfluidic chip to form a segmented stream in which the sample is split into tens of thousands of droplets that act as a PCR chamber and are separated by immiscible liquids (e.g., mineral oil) to form an emulsion [3]. The emulsion is collected in vials, subjected to PCR, and the samples are subsequently processed by flow cytometry to count the number of PCR-positive droplets. or the emulsion is fed into a plastic chip to form a monolayer of droplets, which are thermally cycled and fluorescence images of the droplets are captured and evaluated [4]. Compared with qPCR, ddPCR has the advantage of higher accuracy and lower coefficient of variation for absolute quantification [5]. PCR multiplexing is typically performed by probe-based fluorescence, with a different excitation color for each DNA/RNA. It is also used to generate libraries for single-cell RNA sequencing.

 

Fig.4 Schematic diagram of ddPCR[6]

References:

[1] Jalali M, Zaborowska J, Jalali M. The polymerase chain reaction: PCR, qPCR, and RT-PCR[M]//Basic science methods for clinical researchers. Academic Press, 2017: 1-18.

[2] Zhang H, Tang K, Wang B, Duan CG, Lang Z, Zhu JK. Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants. Plant Methods. 2014 Jun 14; 10:18. doi: 10.1186/1746-4811-10-18. PMID: 24955108; PMCID: PMC4065543.

[3] Schiffman M, Bauer H, Lorincz A et al. Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA. J. Clin. Microbiol.29(3), 573–577 (1991).

[4] Madic J, Zocevic A, Senlis V et al. Three-color crystal digital PCR. Biomol. Detect. Quant. 10, 34–46 (2016).

[5] Hindson CM, Chevillet JR, Briggs HA et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat. Methods 10, 1003 (2013).

[6] Hwang B, Lee JH, Bang D. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp Mol Med. 2018 Aug 7; 50(8):1-14. doi: 10.1038/s12276-018-0071-8. Erratum in: Exp Mol Med. 2021 May; 53(5):1005. doi: 10.1038/s12276-021-00615-w. PMID: 30089861; PMCID: PMC6082860.

 

Catalog number	name	specification
abs60036	2×Taq PCR Mix	1mL×5
abs60037	2×Taq PCR Master Mix(for PAGE)	1mL×5
abs60034	2× Xerox PCR Master Mix	1mL×5
abs60057	2×HotStart Taq plus Master Mix(Quick Load)	1mL×5
abs60056	2×Pfu Master Mix	1mL×5
abs60035	2×Long Taq PCR Master Mix	1mL×5
abs60070	Whole Genome Amplification Kit	50T
abs60246	Reverse transcription is a tube of three generations of master mix	100T
abs601510	First-strand cDNA Synthesis Mix With gDNA Remover	100T
abs60074	One-Step RT-PCR Kit	200T
abs60267	miRNA probe reverse transcription kit	40T
abs60245	Degenomic and reverse transcription in one tube of three generations of master mix	100T
abs60265	miRNA tailing reverse transcription kit	40T
abs60266	Plasma miRNA tailing reverse transcription kit	50T
abs60269	miRNA stem-loop reverse transcription kit	50T
abs60150	RNase inhibitors	1mL
abs60075	Two-Step RT-PCR Kit	1kit
abs601511	2× Premixed real-time PCR reaction system	1.7mL×3
abs60345	SYBR One-Step qRT-PCR Kit	100T
abs60247	Antibody Dye Quantitative PCR Master Mix (Universal ROX)	1mL×5
abs601512	2× Mixed Real-Time PCR Reaction System (High Concentration ROX)	1.7mL×3
abs60086	SYBR High-Sensitivity qPCR SuperMix	1mL×5
abs60270	miRNA stem-loop dye PCR kit	200T
abs601513	2× Premixed Real-Time PCR Reaction System (Low Concentration ROX)	1.7mL×3
abs60272	miRNA probe-based real-time PCR reagents	200T
abs60271	miRNA plus tail dye method real-time PCR kit	200T

 

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