Real Time PCR Principle, Protocol, Application, Advantages.

What is Real-Time PCR?

A real-time polymerase chain reaction (real-time PCR) is a conventional polymerase chain reaction (PCR) based laboratory technique used in molecular biology for real-time monitoring of the amplification of a targeted DNA molecule during the PCR, not at its end, as the conventional PCR do.

It can be used both quantitatively (quantitative real-time PCR) and semi-quantitatively (semi-quantitative real-time PCR).

In real-time PCR the detection of PCR products can be done by two methods such as; (a) by using non-specific fluorescent dyes which will intercalate with the double-stranded DNA, (b) by using fluorescent reporter labeled sequence-specific DNA probes which will detect the PCR product after the hybridization with its complementary sequence.
The conventional PCR detects the amplified DNA product or amplicon at the end of the analysis, while the real-time PCR measures the accumulation of amplification product as the reaction progresses, in real-time, with product quantification after each cycle.

In Real-Time PCR, first amplification reactions are set up with PCR reagents and unique or custom primers. Reactions are then run in real-time PCR instruments and the collected data is analyzed by proprietary instrument software.
Real-time PCR is also known as quantitative polymerase chain reaction (qPCR).
Real-time PCR was Discovered by Kary B. Mullis in the early eighties.

Real Time pcr principle

Real-time polymerase chain reaction is a molecular biology based laboratory technique for amplification and simultaneous quantification of a targeted DNA. This specialized reaction follows the principles of regular PCR in ‘real time’ instead of detecting the end product at the end. For this reason it is called quantitative PCR which identifies the products using either a fluorescent dye or a probe.

The Real-time PCR or Quantitative PCR is a modified approach compared to standard PCR, where the amplified DNA is detected as the reaction progresses. Quantitative PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorophore.

Real-Time PCR Protocol

It is a valuable tool, use for evaluating gene expression by estimating the abundance of certain RNAs. There are two methods are available for Real-Time PCR such as; one-step RT-qPCR and two-step RT-qPCR.

One-Step RT-qPCR

In this method, a single reaction vessel and a common buffer is used to carry out the synthesis of cDNA and qPCR.

This method can be used when you have a lot of sample, and/or a limited number of known targets that you may be repeatedly amplifying.
In One-Step RT-qPCR Gene-specific primers are used which is anneal at higher temperatures than random primers. Sometimes this method uses higher RT reaction temperatures as compared to the two-step workflows and uses engineered or novel RTs that can endure more eminent reaction temperatures.

Advantage of One-Step RT-qPCR

Certainly easier to set up with less overall hands-on time.

It has less pipetting steps which will reduce errors and contamination.
Improved data reproducibility.
For high-throughput applications.

Minimal sample handling, which reduced the bench time.
Closed-tube reactions.
Fast and highly reproducible

Disadvantages

Required new RNA sample(s) to examine new targets or repeat experiments.
Detection of fewer targets per sample
Impossible to optimize the two reactions separately

Less sensitive than two-step because the reaction conditions are a compromise between the two combined reactions

Standard Protocol of One-Step RT-qPCR

Mix the following components, except RNA, in sterile RNase-free microfuge tubes. OneTaq One-Step Reaction Mix (2X) 25 μl, OneTaq One-Step Enzyme Mix (25X) 2 μl , Gene-specific Forward Primer (10 μM) 2 μl, Gene-specific Reverse Primer (10 μM) 2 μl, Nuclease-free H2O 19–x μl, Total RNA (up to 1 μg) x μl, and final volume will be 50 μl.
Add RNA template last, and start reactions immediately, as follows:

CYCLE STEP	TEMPERATURE	TIME	CYCLES
Reverse Transcription	48°C	15–30 minutes	1
Initial Denaturation	94°C	1 minute	1
Denaturation Annealing Extension	94°C 50–65°C 68°C	15 seconds 30 seconds 1 minute per kb	40
Final Extension	68°C	5 minutes	1
Hold	4°C	∞	1

Two-step RT-qPCR

In this method, random hexamers are used to carry out the synthesis of cDNA. Some random hexamers are oligo-dT primers, and/or gene-specific primers.
This method is used when your specimen is precious and/or you need to examine multiple targets or targets that may not be well characterized and may require you to go back to test new targets.
The produced cDNA is is a mixture of various RNA species in the sample.

In this method, the cDNA synthesis reaction can be scaled up to provide higher RNA input, and extraction and precipitation steps can be used to concentrate and/or further purify the cDNA.
One portion of the cDNA produce is used in the following qPCR step, resting cDNA can be saved for future use, or quantitating the expression of multiple genes from a single RNA/cDNA sample.

Advantages

The same RNA sample can be used for multiple targets due to separate reactions.

Provide Greater flexibility to select RT enzymes and DNA polymerases for PCR separately.
Store cDNA for later use.
The approved method for applications with a limited amount of starting material (i.e. single cell analysis)

Disadvantages

It requires an extra open-tube step.
More pipetting manipulations which may lead to greater variability and risk of contamination.
Longer hands-on time.

Protocol of Two-step RT-qPCR

Step One: Reverse Transcription

Reagents Required

Reverse Transcriptase MMLV-RT (100 units/µl)
10X RT Buffer (500 mM Tris-HCl, pH 8.3, 750 mM KCl, 30mM MgCl2 50 mM DTT)

Random decamers (50µM)
dNTP (2.5mM each dNTP)
RNase Inhibitor (10 units/µl)

Place RNase Inhibitor and Reverse Transcriptase ON ICE directly from the Box. Thaw 10x reaction buffer, random decamers, and dNTP mix quickly in your hands and place ON ICE;

Procedure

Use small 0.25ml PCR tubes

Assemble your reaction as follows on ice. Add the enzyme last.

Real-Time PCR Principle, Protocol, Application, Advantages.

Mix gently, spin briefly.
Incubate in the thermocycler at: a. 44°C for 1 hr; b. 92°C for 10 min to inactivate the reverse transcriptase.

Store reaction at –20°C or proceed to the PCR.

Step Two: PCR

A. Primer Preparation

Primers are shipped in dry form. Briefly spin the tube before you open the cap to avoid loss of DNA pellet.
Dissolve the oligonucleotide in 10mM Tris, pH7.5 to make a primer stock at 100µM concentration.

Dilute from this stock 1:20 (in water) to make a working solution at 5 µM for use in setting up PCR reactions.

B. Setting up PCR reactions

Negative Controls

Use two negative controls among the PCRs.

The minus-RT control from the previous step, or alternatively, untreated RNA can simply be subjected to PCR.
A minus-template PCR, should have all the PCR components but use water as a template instead of an aliquot of the cDNA (RT reaction). This control will verify that none of the PCR reagents is contaminated with DNA.

Positive Control

Perform PCR to amplify a cDNA that corresponds to a basal/housekeeping transcript, e.g., ribosomal protein S17 (RpS17). The TA’s will provide each team with RpS17 primers. For your reference, these are the primer sequences: Forward RpS17: 5′ – cga acc aag acg gtg aag aag – 3′ Reverse RpS17: 5′ – cct gca act tga tgg aga tac c – 3′

Expected RT-PCR product size: 211bp .The primers are located on different exons that are separated by a 59bp intron. If genomic DNA is amplified, the product size would be 270bp.

Use genomic DNA isolated from S2 cells as template. If your primers span intron(s), note the size of the expected PCR product and if necessary, adjust annealing temperature of the PCR program.

Reagents:

Taq DNA polymerase: Invitrogen
With 10X Buffer (-MgCl2) and 50mM MgCl2

The following table outlines the components needed for PCR. Note: do not use DEPC-treated water.

For RpS17 control:

Making Master Mixes:

Consider making master mixes if you are testing multiple sets of primers at once. A master mix will contain everything except the PCR primers. If you are testing n sets of primers, make a master mix enough for n+1 tests.
Mix the components gently but thoroughly. Aliquot 22.5µl of your master mix to each tube.
Add 1.25µl of each of the appropriate primers at 5µM working stock concentration.

Assemble reactions on ice.

Incubate in Thermacycler:

Initial denaturation: 94°C for 4 min

30 cycles: Denature at 94°C for 30 sec, Anneal at 55°C for 20–30 sec**, Extend at 72°C for 45 sec ***
Final extension: 72°C for 5 min

Note: Start with the annealing temperature suggested by your primer design software. An annealing temperature of ~55°C used with the cycling times shown is often a reasonable starting point, but the optimal temperature and cycling times for your primer and template combination may need to be determined empirically.

Note: The rule of thumb is to use an extension time of 1 min per kilobase of the target.

Run 15 µl of your reaction on 1-1.5% Agarose gel

one-step RT-qPCR and two-step RT-qPCR — Image source https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/basic-principles-rt-qpcr.html

Fluorescence Markers for Real-Time PCR

The most commonly used Fluorescence Markers are;

Taqman Probe

Type of hydrolysis probe, at the 5’ end it contain a reporter dye, often fluorescein (FAM) and a quencher tetramethylrhodamine (TAMRA), connected to the 3’ end of the oligonucleotide.
Under standard conditions, the probe rest folded on itself taking the fluorescence dye near the quencher, which hinders of the fluorescent sign of the dye.
The Taq polymerase oligonucleotide contains a homologous region with the target gene as the target sequence is present in the mixture, it binds with the sample DNA.

During the extension stage, the Taq polymerase induces degradation of the probe by 5’ end nuclease action and the fluorescein is segregated from the quencher as it leads to the formation of fluorescence.
The number of signal molecules gradually increases in each cycle, as it results in the increase in fluorescence which is positively related to the amplification of the target.

SYBR Green

On binding to the minor groove of DNA, it emits a prominent fluorescent signal.

The SYBR Green has a higher signal intensity as compared to other fluorescent dyes such as Ethidium Bromide or Acridine Orange.
The SYBR Green can provide data about each cycle of amplification and also about the melting temperature, that’s why it is a more preferable marker as compared to Taqman Probe.
The lack of specificity is one of the disadvantages of SYBR Green as compared to Taqman Probe.

Advantages of Real-Time PCR

The wide dynamic range of quantification (7-8 logarithmic decades)
High analytical sensitivity (<5 copies; 1fg to 10 pg bacterial DNA per PCR reaction)
Better precision (<2% standard deviation)

Closed system to reduce risk of contamination
No post PCR processing
Lower turnaround time

Increased throughput
Multiplexing capabilities

Disadvantages of Real-Time PCR

PCR product increases exponentially cannot monitor amplicon size.

Variation increase with cycle number
Maximum of four simultaneous reactions
Overlap of emission spectra

Increased risk of false-negative in pathogen detection (particularly for new emerging or highly variable pathogens)
Non-specific binding (SYBR green analysis)

Application of Real-Time PCR

There are the different application of real-time PCR such as;

Quantification of gene expression: The quantification of nucleic acids is doen by two methods: relative quantification and absolute quantification. Absolute quantification provides the specific number of target DNA fragments by association with DNA standards utilizing a calibration curve. Relative quantification is based on internal reference genes to discover fold differences in the expression of the target gene.
Diagnostic uses: Used for diagnosis of infectious diseases, cancer, genetic abnormalities, and also used for diagnoses of emerging diseases, such as new strains of flu and coronavirus.
Microbiological uses: Microbiologists uses this in working fields of food safety, food spoilage, and fermentation and also in water quality maintenance.

Detection of phytopathogens: Real-Time PCR also used for the detection of phytopathogens.
Detection of genetically modified organisms: qPCR using reverse transcription (RT-qPCR) can be used to detect GMOs given its sensitivity and dynamic range in detecting DNA.
Clinical quantification and genotyping: This helps in both the quantification and genotyping of a virus such as the Hepatitis B virus.

References

International Journal of Multidisciplinary and Current Research Detection of Water-borne Pathogenic Bacteria: Where Molecular Methods Rule – Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Advantages-and-Limitations-of-Real-Time-PCR_tbl2_292318147 [accessed 27 May, 2021]
https://international.neb.com/protocols/2014/08/15/one-step-rt-pcr-protocols-e5315
https://ocw.mit.edu/courses/biology/7-16-experimental-molecular-biology-biotechnology-ii-spring-2005/labs/rt_pcr_2step.pdf

https://microbenotes.com/real-time-pcr-principle-process-markers-advantages-applications/
https://www.thermofisher.com/in/en/home/brands/invitrogen/molecular-biology-technologies/spotlight-articles/onestep-vs-twostep-rtpcr.html
https://himedialabs.com/TD/HTBM032.pdf