Real-Time PCR Analysis
Polymerase Chain Reaction (PCR) is one of the most accurate methods for analyzing gene expression. This means it can determine whether the expression of specific genes in a cell will increase, decrease, or remain unchanged under certain conditions. Before the advent of Real-Time PCR, the most common methods for determining gene expression levels included Northern blotting, RNase protection assays, and endpoint reverse transcription (RT) PCR. RT-PCR was more widely used than the other two methods due to its convenience and lower RNA requirements. However, this method only allows for observation of gene expression levels at the end of the reaction through electrophoresis of the PCR product on an agarose gel. Therefore, RT-PCR is mainly useful for determining the presence or absence of a specific gene. In fact, this test is qualitative or semi-quantitative, and thus cannot provide a quantitative analysis of a specific gene expression. Consequently, there was a need for a method that is entirely quantitative.
Real-Time PCR is essentially the same as PCR, with the distinction that in Real-Time PCR, the amplification and increase in the number of RNA copies during the reaction is measured using fluorescent materials. This method utilizes substances that indicate when each cycle of the reaction occurs, allowing for precise and quantitative analysis of gene expression. Given the aforementioned points, it is essential to have a control (meaning a cell without any applied changes) when performing this test.
Advantages of Real-Time PCR:
Measurement of the number of RNA copies of a gene at the starting phase and determination of the smallest differences in expression levels between samples.
Observation of the gradual increase in PCR products throughout the experiment.
No need for agarose gel electrophoresis, eliminating the risk of contamination at this stage.
Materials Required for the Test:
Total RNA extraction kit
cDNA synthesis kit
Master Mix for Real-Time PCR
Thermo cycler
Steps of Real-Time PCR Testing
RNA Extraction
The first step in conducting Real-Time PCR is RNA extraction, which varies based on the type of sample. Generally, RNA extraction can be performed in two ways:
Organic extraction methods using reagents like Trizol, Qiazol, RNA STAT-60T, and methods based on guanidine salt.
Various commercially available solid-phase RNA isolation kits from different companies.
It is crucial that the RNA extracted from all samples is done using a uniform method. If the extracted RNA is not to be tested immediately, it should be stored at -20°C for up to one month, and for longer durations at -80°C. The absorbance ratio at wavelengths 260/280 of the extracted RNA can indicate the quality of the extraction; a ratio of 1.8 indicates good RNA extraction quality.
Reverse Transcription
To perform Real-Time PCR, it is necessary to synthesize cDNA from RNA. Various kits have been developed for this purpose, all based on the same principle. All these kits utilize the enzyme MMLV, which is a reverse transcriptase. Other materials used for producing cDNA include random hexamers or Oligo dT, which act as primers for the relevant enzyme. After adding the above materials at the concentration specified by the manufacturer, the reaction is placed in the PCR device at a specified temperature and time, which is also determined by the manufacturer. It is essential that the RNA from all samples used for preparing cDNA is of equal quantity, as this test should demonstrate the increase in expression of a specific mRNA at uniform RNA amounts.
Internal Control
In conducting Real-Time PCR, in addition to the target genes for determining expression levels, another gene must also be included as a housekeeping gene. The role of this gene is to indicate the accuracy of cDNA synthesis, the purity of the obtained RNA, the correctness of the PCR test, and to normalize the expression of other genes. Therefore, before starting any procedure, primers must first be designed (or sourced from articles) and synthesized for all genes to be tested. The following materials are essential in every PCR reaction: DNA polymerase enzyme (known as Taq), MgCl2 buffer, dNTPs, primers, and finally cDNA.
Negative Control
It is important to note that for every gene tested, an additional reaction containing all the materials present in the PCR reaction except for cDNA will be conducted. The necessity for this additional reaction is to check for the absence of contamination in the samples and to ensure there are no reactions such as primer dimers in the test.
Sources of Error:
Incorrect RNA extraction
Insufficient RNA purity
Failure to synthesize cDNA
Errors in adding PCR materials
Non-calibrated PCR device
Contamination during any stage of the process
Poorly designed and specific primers
Presence of cellular DNA contamination
Quantitative Analyses in Real-Time PCR
There are two main methods for quantitative assessment in Real-Time PCR:
1- Standard Curve Method (Absolute Comparison):
In this method, a sample of RNA or DNA with a known concentration is used to create a standard curve. The concentration of the standard RNA or DNA is determined using a spectrophotometer (nm260), and then converted to the number of copies based on the sample's molecular weight. Concentration standards for well-known genes are commercially available, although they can be quite expensive. A series of dilutions of the standard samples is prepared and placed alongside the target sample in the Real-Time PCR device. Using the Ct values provided by the device for each dilution, a curve is plotted where X represents the dilution or number of copies of the gene and Y represents the Ct. The resulting graph is a linear graph that allows for determining the concentration or number of copies of the target sample by placing its value on the graph. Ideally, the length of the standard fragment should be equal to that of the target fragment.
2- Relative Threshold Method (Relative Comparison):
To eliminate fluctuations in the amounts of RNA introduced into the reaction and operational errors of devices and personnel, internal standards are used. These standards should have a stable expression across all tissues, and our assay should not alter their expression compared to the control sample. For this purpose, the genes Beta-actin and GAPDH are utilized, referred to as housekeeping genes. The expression of these genes is stable, allowing for the observation of increases or decreases in expression by comparing the treated sample with the control and the internal standard.
Detection of Polymorphism by Real-Time PCR
The use of this technique allows for quantifying regions of DNA polymorphism or determining the genotype of a new single nucleotide polymorphism (SNP). Simultaneous monitoring of the amplification process and genotype determination is possible through probe hybridization and melt curve analysis. Point mutations can be detected through melt curve analysis. The critical temperature (Tm) at which DNA becomes single-stranded serves as an indicator for the presence or absence of a mutation. A specific probe for the mutant region is prepared; at lower temperatures, the probe binds to non-specific regions, and as the temperature increases, the probe becomes more specific. The resulting melt curve is analyzed to determine the presence or absence of the mutation.
