1. Distinction Between RT-PCR, qPCR, Real-time PCR, RT-qPCR
RT-PCR refers to Reverse Transcription PCR, in which RNA is reverse transcribed to cDNA and DNA is amplified by PCR using cDNA as a template.
qPCR and Real-time PCR both refer to Quantitative Real-Time PCR. qPCR enables real-time quantification of amplification products in each cycle compared to conventional PCR, resulting in precise analysis of the amount of starting template with high sensitivity and specificity.
RT-qPCR refers to Reverse Transcription Quantitative PCR), which is a combination of qPCR + RT-PCR that reverse transcribes mRNA to cDNA before using it as a template for qPCR quantitative analysis.
2. SYBR Green I Dye Method
SYBR Green I dye: a highly specific double-stranded DNA binding dye that can detect PCR products as they accumulate during the PCR cycles.
Principle:
Real-time detection of PCR products by binding SYBR Green I dye to double-stranded DNA produced during PCR.
Process:
(1) During PCR, DNA polymerase amplifies the target sequence to generate PCR products, i.e. "amplicons";
(2) SYBR Green I dye binds to each newly generated double-stranded DNA molecule;
(3) As PCR progresses, more and more amplicons are generated, and since the SYBR Green I dye binds to all double-stranded DNA, and therefore the fluorescence intensity increases with the PCR product.
Advantages:
No selectivity for DNA template, can be used to monitor amplification of any double-stranded DNA sequence, low running costs.
Disadvantages:
SYBR Green I dye may bind to non-specific double-stranded DNA sequences, generating false positive signals. In addition to this, constant optimization of the reaction system is required to reduce non-specific amplification.
3. Taqman Probe Method
Principle:
Detection of PCR products by accumulation of fluorescent probes with them during the PCR cycle.
Process.:
(1) Construct Oligo probe: 5' end is labeled with fluorescent dye reporter group (Reporter, R) and 3' end is labeled with quenching group (Quencher, Q). (1) When the probe is left intact, the proximity of the quenching group significantly reduces the fluorescence emitted by the reporter group through spatial fluorescence resonance energy transfer (FRET).
(2) If a target sequence is present, the probe then anneals downstream of one of the primer binding sites and completes excision as the primer is extended through the 5' nuclease activity of Taq DNA polymerase.
(3) The excision of the probe separates the reporter dye moiety from the quenched dye moiety, enhancing the signal of the reporter dye moiety. and allows the probe to be removed from the target strand, allowing the primer to continue to extend along the end of the template strand.
(4) With each cycle, more reporter dye molecules are cut off from their respective probes, and the fluorescence intensity increases with the number of amplification fragments synthesized.
Advantages: (compared to SYBR Green I dye method)
(1) High specificity and sensitivity: the fluorescent probes bind only to the target sequence and are not affected by non-specific products.
(2) Compatible with multiplex reactions: since different wavelengths of fluorescent reporter groups can label different probes, different reporter gene dyes can be selected to label the probes, and different sequences can be amplified and detected simultaneously in one reaction tube.
(3) Time and material cost savings: no PCR post-processing is required, reducing analytical workload and saving material costs.
4. Absolute Quantification
Principle:
The quantification of an unknown sample based on a standard curve.
Example:
The initial copy number of the sample to be tested can be calculated by qPCR, such as how many HBV viruses are in 1 ml of blood. The disease status can be monitored based on the copy number of the virus.
5. Relative Quantification
Principle:
Used to analyze the change in expression of a gene in a specific sample relative to a reference sample.
Example:
To detect drug-induced changes in gene expression, the expression level of a specific target gene in a drug-treated sample is compared to the expression level of a gene in an untreated sample.
Professional Portable qPCR System Manufacturer - Anitoa
Anitoa is a professional R&D and manufacturing company of molecular diagnostic qPCR instruments and reagents, with independent intellectual property rights of core chip technology, optical technology, rapid heating, microfluidic control and supporting reagents and other patented technologies, so that the instruments developed by Anitoa are characterized by quick and easy small instruments, allowing the development of expensive and complex large PCR instruments into truly portable POCT products.