PCR, RT-PCR and qPCR Protocol: Amplification and Analysis

PCR, RT-PCR and qPCR

Polymerase Chain Reaction (PCR), Reverse Transcription-Polymerase PCR (RT-PCR) and quantitative PCR (qPCR)

Overview

Polymerase chain reaction (PCR) is a technique applied for amplification and quantification of DNA. In the PCR process, the ds-DNA in the sample first undergoes denaturation, and the double-stranded DNA is separated. Then, in the process called annealing, primers, the short DNA fragments, bind to the complementary sequence of DNA, and provide a starting site for DNA polymerase. Finally is the extension, DNA polymerase synthesis of new DNA strands, and forms two ds-DNA molecules. As a result, specific DNA fragments are exponentially amplified.

In reverse transcription polymerase chain reaction (RT-PCR), an RNA population is converted to cDNA by reverse transcription (RT), and then the cDNA is amplified by the PCR. In quantitative PCR (qPCR), PCR amplification is combined with a fluorescent probe. By monitoring fluorescent signals, the content of specific DNA fragments is quantitatively determined in real time.

Material and Equipment

• PCR tubes
• Thermal cycler: control temperature for reverse transcription of cDNA.
• Conventional PCR: Agarose, Electrophoresis buffer (TAE or TBE), DNA dye

(e.g., EtBr or safety dye), DNA ladder (molecular weight standard), Loading buffer

• Rotary real-time quantitative polymerase chain reaction (qPCR)

instrument: quantitative nucleic acid analysis in molecular biology and clinical research, e.g. Qiagen Rotor-Gene Q.

Reagent

• DNA polymerase: catalyses the synthesis of new DNA strands during

cDNA synthesis and DNA amplification in PCR, such as M-MLV reverse transcriptase (RNA-dependent DNA polymerase for reverse transcriptase)

and Taq polymerase (DNA-dependent DNA polymerase for PCR).

• Deoxynucleotide triphosphates (dNTPs): consist of deoxyadenosine

triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP), each corresponding to one of the four DNA bases. During the reverse transcription of cDNA and the extension phase of PCR, DNA polymerase incorporates dNTPs into the growing DNA strand by matching them with complementary bases on the template strand. In this process, dNTPs supply both nucleotide and chemical energy necessary for DNA polymerisation.

• Random primers: Random primer are a group of short DNA sequences,

composed of multiple different nucleotides, without specific sequence characteristics. The random primer contains short DNA sequences, randomly combined with RNA, to initiate the process of DNA synthesis.

• Dithiothreitol (DTT): a reducing agent maintains the reverse transcriptase

system in a reduced and active state by preventing the formation of disulfide bonds, thereby preserving enzyme structure and ensuring efficient cDNA synthesis.

• Mg²⁺ ions: a cofactor for cDNA synthesis enzyme activity.
• Buffer system: maintains optimal pH and ionic conditions for efficient and

specific amplification. DNA-binding fluorescent dye: dye specifically binds to double-stranded DNA, e.g. SYBR Green dye. During PCR amplification, as the amount of double-stranded DNA increases, the fluorescence intensity correspondingly increases, directly reflecting the accumulation of PCR products.

• Forward primer and reverse primer: forward primer and reverse primer are

a pair of short DNA fragments, which bind to the two ends of the target DNA respectively to provide a starting point for DNA polymerase amplification. The forward primer binds to the 5' end of the template DNA and is complementary to the positive strand of the target DNA, and the reverse primer binds to the 3' end of the template DNA and is complementary to the reverse strand of the target DNA. Through the design of this pair of primers, PCR can specifically amplify the target DNA region.

• cDNA template: DNA functions as a template for PCR amplification,

including housekeeping genes (e.g. actin or 18s) and target genes.

• RNase-free water: Ultrapure water free from RNase contamination, used

for dilution of reagents while preventing RNA degradation during cDNA preparation.

• ROX reference dye: passive reference fluorescent dye, which generates a

fluorescence signal that does not change with PCR. It can be used for signal correction and eliminating batch differences between different samples, volume differences between reaction tubes and fluctuations in fluorescence signal intensity, improves the accuracy and comparability of PCR data. ROX high and ROX low refers to different concentrations of ROX dye, suitable for instruments that require different fluorescence signals, and selected based on the product brochure from the manufacturer.

Reagents essential required for RT-PCR assay are often provided as a pre-formulated reaction solution in a commercial product, e.g. KAPA SYBR FAST qPCR Master Mix. This master mix supports both efficient DNA amplification and quantitative fluorescence-based measurement in PCR experiments.

Overview of the procedure

Reverse Transcription

1. Add 1 μg of RNA into a tube, and then add RNase-free water to form an 11

μl solution.

1. Add random primers (1 μl 50 μg/ml) and dNTPs (1 μl 10 mM) to each

sample, then incubate the mixture solution at 65 °C for 5 min.

1. Add buffer (e.g. 5X first-strand buffer) (4 μl) and Dithiothreitol (DTT) (2 μl),

then incubate the mixture solution at 37 °C for 2 min.

1. Add M-MLV reverse transcriptase (1 μl) and RNase-free water (1 μl) , then

incubate the mixture solution at 25 °C for 10 min, incubate at 37 °C for 50 min, and then incubate at 70 °C for 15 min to inactivate the reaction.

Conventional PCR assay

1. All reagents are thawed on ice and mixed gently before use. The PCR reaction

mixture is prepared on ice by combining template DNA, forward and reverse primers, dNTP mix, DNA polymerase (e.g., Taq DNA polymerase), PCR buffer (with Mg²⁺ or supplemented with MgCl₂), and nuclease-free water to achieve the desired final volume. The mixture is gently mixed and briefly centrifuged to collect all components at the bottom of the tube.

1. The prepared reaction mixture is dispensed into PCR tubes or a PCR plate. A

negative control containing all components except template DNA is included to detect contamination. A positive control may also be included to confirm that the reaction conditions are suitable for amplification.

1. The PCR tubes are placed into a Thermal Cycler, and the appropriate cycling

program is set. An initial denaturation step is performed at 94–95 °C for 2–5 minutes to fully separate the DNA strands.

The reaction then undergoes 25–35 amplification cycles, each consisting of denaturation at 94–95 °C for 15–30 seconds, annealing at 50–65 °C for 15–30 seconds depending on the primer melting temperature, and extension at 72 °C for 30–60 seconds.

A final extension step is carried out at 72 °C for 5–10 minutes to ensure complete synthesis of all DNA strands.

The reaction is then held at 4 °C until further analysis.

1. The amplified PCR products are analyzed by Agarose Gel Electrophoresis.

An agarose gel is prepared, and PCR products are mixed with loading buffer and loaded into the wells along with a DNA ladder. Electrophoresis is performed, and DNA bands are visualized under UV or blue light.

1. The presence of DNA bands at the expected size indicates successful

amplification of the target sequence. The band sizes are compared with the DNA ladder to confirm the correct product length.

The negative control is checked to ensure that no contamination has occurred, and the positive control is examined to verify that the reaction conditions were effective.

qPCR Assay

1. The primer mix is prepared by mixing the forward primer, reverse primer and

RNase-free water in a ratio of 1:1:18.

1. Mix the pre-formulated reaction solution, the primer mix, ROX High (ROX

reference dye) and water in a ratio of 125:20:1:5 to prepare a master mix of PCR primer solution.

1. In the PCR reaction tube for each sample, 2 μl of cDNA sample and 3 μl

master mix of PCR primer solution is mixed, then the tubes are capped and loaded into the PCR Instrument.

The qPCR protocol used conforms to the following parameters:

Table 1 The parameters set for the PCR process

step Temperature Duration Cycles Enzyme activation 95 °C 3 min Hold Denaturation 96 °C 1 -3 sec 40

Annealing/ extension/ data

acquisition

60 °C 20 sec The assay is repeated separately three times for each sample and primer.

Conventional PCR Results Analysis

The size of the PCR product is determined by comparing the position of the sample band to the DNA ladder. A single clear band at the expected size indicates successful and specific amplification of the target sequence.

No-template control (NTC) should show no bands.

qPCR Results Analysis

Q-PCR produced the cycle threshold (CT) value, also known as the take-off value, which is a dimensionless value that represents the cycle threshold at which the target gene is detected. This threshold denotes the cycle count at which the fluorescence signal first surpasses the background level during PCR amplification.

A reduced CT value indicates an elevated starting quantity of the target gene in the sample. The CT value can be used to compare the abundance of target genes in several samples, or the expression levels of different genes in the same sample.

CT values can be converted into relative or absolute quantitative gene expression levels by constructing a standard curve or using relative gene expression methods, such as the 2^-ΔΔCT analysis method. However, the absolute size of the CT value does not directly reflect the absolute expression level of the gene. In this assay, the CT values produced in RT-PCR assays are analyzed through the 2^-ΔΔCt method.

ΔCt (target gene) = Ct (target gene) - average Ct (housekeeping gene)

The ΔCt value refers to the difference between the take-off values of a target gene and the housekeeping gene for the same sample. Through the step from CT to ΔCt, the results of the housekeeping gene are used as the control group for the individual gene expression level, which is subtracted to eliminate this difference.

ΔΔCt (each group) = ΔCt (each group) - ΔCt (negative control)

The ΔΔCt value refers to the difference between the ΔCt values of each group and their negative control group of the same primer. Eventually, the 2^-ΔΔCt is produced by 2 to the power of a negative “ΔΔCt value” and produces a relative fold of gene expression in all the groups to the negative control group.

The polymerase chain reaction is characterized by an exponential process, wherein the specifically amplified product ideally doubles with each cycle. The measured Ct (cycle threshold) value is a logarithmic measurement that requires conversion into a linear relative quantity. The average Ct is computed for both the target genes and the housekeeping gene, with the ΔCt (delta threshold) defined as the mean of the triplicate Ct values for the target gene subtracted by the mean of the triplicate.

Ct values for Actin. The ΔΔCT (delta delta threshold) indicates the variation between samples. The gene of interest's expression levels are normalized by dividing by the relative expression level of the housekeeping gene for the corresponding sample. The fold-change in gene expression is determined by dividing the normalized expression level of the experimental sample by that of the control sample. Extreme values are excluded.

Application

1. Gene expression: RT-PCR is widely applied to analyse gene expression by

converting RNA in the sample into cDNA, followed by amplification. Compared with the housekeeping gene, the expression of the target gene can be measured.

The impact of bioactive compounds on a specific gene expression can be evaluated by comparing the test and negative control groups.

1. RNA virus detection: Viral RNA is first reverse-transcribed into cDNA,

which is then amplified for detection. This allows for highly sensitive and specific identification of viral infections, as well as quantification of viral load. For example, RT-PCR has been extensively used as a gold standard method for detecting COVID-19.

1. Validating high-throughput data: High-throughput methods, such as

RNA-seq or microarray analysis, allow for the simultaneous analysis of thousands of genes, but these methods may introduce variability or false positives.

RT-qPCR, with its high sensitivity and specificity, is therefore employed to confirm the expression levels of selected genes, ensuring the accuracy and reliability of the overall results.

Limitation

• In RT-PCR, RNA is first reverse transcribed into cDNA prior to amplification,

but this step can introduce additional variability because cDNA synthesis efficiency may differ across samples due to factors such as enzyme performance and primer choice, potentially leading to inaccurate gene expression measurements.

• In conventional PCR, DNA amplification is assessed at the plateau phase

using agarose gel electrophoresis, whereas qPCR enables real-time monitoring of DNA amplification during the exponential phase, resulting in higher sensitivity and reduced risk of operational error.

• The accuracy of qPCR depends heavily on amplification efficiency, which

ideally should result in a doubling of target DNA in each cycle to ensure a consistent and predictable amplification rate.

• In practice, amplification efficiency can vary between samples or targets

due to factors such as primer design, reaction conditions, and template quality, which may distort Ct values and reduce the reliability of comparative analyses such as the ΔΔCt method, ultimately affecting the accuracy of gene expression quantification.

Troubleshooting

Closing statement

RT-PCR serves as a fundamental technique for gene expression analysis and RNA virus detection by converting RNA into cDNA via reverse transcription and exponentially amplifying target sequences. While conventional PCR provides qualitative results through agarose gel electrophoresis, quantitative PCR (qPCR)

enables precise real-time monitoring of DNA accumulation using fluorescent dyes and the 2^-ΔΔCt normalization method. Despite its high sensitivity and widespread application as a gold standard in molecular biology, the accuracy of the results depends heavily on maintaining an RNase-free environment and ensuring consistent amplification efficiency across all samples.