Quantitative Reverse Transcription PCR (RT-qPCR) is a powerful method that adds fluorescent tags to the PCR reaction system. It enables real-time monitoring of the entire PCR process through the accumulation of fluorescence signals, and quantitative analysis of the starting template. This method involves the extraction of RNA from the collected sample, which is then reverse transcribed into complementary DNA (cDNA) using reverse transcriptase. Next, a qPCR reaction is performed using cDNA as a template, and fluorescence signals are collected in real-time to monitor the amplification reaction process.
Currently, RT-qPCR finds extensive use in numerous fields of molecular biology. In scientific research, it can be used for gene expression analysis, pathogen detection, and RNA interference verification. It has also become the gold standard for molecular in vitro diagnosis. It is used in various application scenarios such as clinical disease diagnosis, animal inspection and quarantine, food safety, and scientific research. The technique plays a crucial role in the detection, diagnosis, and treatment of diseases such as the novel coronavirus, hepatitis, AIDS, influenza, dengue fever, and infectious diarrhea.
RT-qPCR Experimental Protocol
Sample Collection
01 Avoiding the Influence of Exogenous RNases
Exogenous RNases are the main factor that causes RNA degradation. To avoid their influence, the collection and experimental environment should be as clean as possible, and consumables should be treated with DEPC or purchased as enzyme-free consumables. Experimenters should also be quick and skilled in their operations to shorten the degradation time.
02 Avoiding the Influence of Endogenous RNases
Certain RNA-rich areas that contain endogenous enzymes, such as the spleen and thymus, are prone to degradation. To prevent this, it is recommended to crush the tissues under liquid nitrogen conditions and use more lysing solution during homogenization.
03 Sample Aliquoting Based on Extraction Amount
Before sampling, the amount of tissue extracted at once should be clearly defined, and the sample should be divided and stored according to the amount extracted at once. This prevents contamination and degradation caused by repeated or combined pipetting.
04 Selecting High-Abundance Tissue Sites
The abundance of RNA varies across different tissue sites, and tissue sites with high abundance should be selected as much as possible. For example, animal liver, spleen, and heart samples can reach an RNA abundance of 2-4 μg/mg, while plant leaf and root samples have an RNA abundance of 0.2-0.3 μg/mg.
Sample Storage
The storage method has a significant impact on the quality of the sample RNA. The appropriate storage method and operation should be selected based on the sample type. Generally, samples are stored at low temperatures using RNA tissue cell storage solution or Trizol. If RNA is not extracted immediately, the sample can be ground and homogenized properly and then quickly frozen with liquid nitrogen. Proper homogenization helps to disperse cells and ensure full contact with the storage solution (infiltration), reducing the degradation of RNA inside the sample. Blood samples can have anticoagulants such as sodium citrate added. Avoid repeated freeze-thaw cycles, as the success rate of RNA extraction from tissue cells significantly decreases after repeated cycles.
RNA Extraction and Purification
There are two commonly used methods for RNA extraction: liquid-phase extraction using guanidine thiocyanate/phenol reagents (such as Trizol-like reagents) and column-based extraction using silica membrane-specific adsorption.
01 Trizol Method
Trizol reagent contains guanidine thiocyanate and phenol as its main components. Guanidine thiocyanate is a denaturant that can dissolve proteins and is used to lyse cells, causing proteins and nucleic acids to disperse and releasing RNA into the solution. Phenol is used to denature proteins, but it cannot completely inhibit the activity of RNases. To address this issue, Trizol also contains components such as 8-hydroxyquinoline and beta-mercaptoethanol to inhibit both endogenous and exogenous RNases. When chloroform is added, it can extract acidic phenol, which promotes RNA to enter the aqueous phase. After centrifugation, the mixture separates into a water phase and an organic phase, with RNA remaining in the aqueous phase and proteins and DNA in the organic phase.
02 Column-based Extraction Method
The column-based extraction method does not require the use of toxic phenol/chloroform for extraction, and it can quickly lyse tissue cells while inhibiting endogenous RNases to protect RNA. Under high ionic strength and low pH conditions, the negatively charged nucleic acid skeleton in RNA binds strongly to the positively charged silica membrane filter. Impurities are removed by washing, and the RNA is then eluted with an elution buffer under low ionic strength and high pH conditions.
Reverse transcription synthesis of cDNA
Generally, RNA is reverse transcribed into cDNA using reverse transcriptase as a template for downstream experiments such as PCR and cloning. Commercially available reverse transcription kits usually include dNTPs, reverse transcriptase, RNase inhibitor, and reverse transcription primers.
Real-time fluorescence quantitative PCR
Real-time fluorescence quantitative PCR (qPCR) is a powerful technique used for the detection and quantification of nucleic acid sequences. When designing primers for qPCR, there are several important considerations to ensure successful amplification and accurate results.
Firstly, intron-spanning primers can be chosen to reduce the influence of genomic DNA. Alternatively, reverse transcription methods that remove gDNA can be used to avoid interference.
The length of the primer should be between 18-30 nt, with the length of the amplified product between 100-300 bp. Primers that are too short can cause non-specific amplification, while those that are too long can form secondary structures, which can affect amplification efficiency.
The GC content of the primer should be controlled between 40-60%, and the upstream and downstream primers should have similar GC content to achieve the same Tm value (55-65℃). It is best to avoid selecting "A" at the 3’ end of the primer, as this can significantly reduce synthesis efficiency in the presence of a mismatch.
For probes, the 5' end should not be "G", as even a single "G" base can quench the fluorescence signal emitted by the FAM group, leading to false-negative results.
The base distribution should be random, and the 3’ end should avoid having more than three consecutive "G" or "C" bases to prevent pairing in GC-rich sequences. Complex secondary structure sequences can affect the successful progress of PCR, so it is important to avoid designing primers in these areas by predicting and analyzing the sequence in advance.
Primer dimerization can be prevented by avoiding continuous four complementary bases between the primer itself and different primers. If primer dimerization and hairpin structures are unavoidable, it is important to keep the ΔG value below 4.5 kcal/mol.
Finally, after designing the primers, a BLAST test should be performed to compare the specificity of the product with related sequences in the database to avoid non-specific amplification of similar but non-target sequences.
Common qPCR Detection Methods
Common qPCR detection methods include real-time fluorescence quantitative PCR, which can be divided into two methods: SYBR Green I fluorescent dye method and Taqman probe method.
SYBR Green I fluorescent dye method uses SYBR Green I as a commonly used dye, which can non-specifically bind to the minor groove of double-stranded DNA but not to single-stranded DNA. Although this lack of specificity means that the dye can bind to both target and non-target genes, the dye produces a strong fluorescence signal once it binds to double-stranded DNA, making it useful for detecting target genes.
TaqMan probe method uses a short nucleotide sequence that is labeled with a fluorescent reporter group at its 5' end and a quencher group at its 3' end. The fluorescence signal generated by the fluorescent group is suppressed by the quencher group when the probe is intact. Once the probe is hydrolyzed, the fluorescent and quencher groups separate, allowing the fluorescence signal to be detected by the instrument.
In general, the probe method is commonly used in molecular diagnostics due to its higher sensitivity and specificity, which allow for more accurate detection. In scientific research, however, the dye method is more commonly used for analyzing gene expression levels.
Experimental Optimization Techniques
01 Solidification strategy and mechanical operation
qPCR often involves a large number of reactions that require the addition of reagents in a dense manner. It is recommended to add samples to a 96-well plate in a consistent manner. First, mix the primers and SYBR green mix into a Master Mix and add it to each column. Then add the diluted cDNA to each row using the same method. Add the samples in the following order for cDNA: control first, followed by the experimental group, and finally the reference (without reverse transcriptase). For primers, place the housekeeping gene in the first column, and then arrange the other primers in alphabetical order. The advantage of this method is that even if a small mistake is made, the position that needs to be added can still be identified.
02 More is Better!
When pipetting, using a larger volume is better (the reason being obvious). As mentioned earlier, start by mixing the Master Mix and adding it to the 96-well plate, followed by adding the diluted cDNA mix (usually diluted 20 times) in the same way. This approach can help avoid using extremely small volumes, which can improve accuracy. Typically, a final volume of 20 μl is used, with 11 μl of Master Mix and 9 μl of cDNA added separately.
03 Repetition, More Repetition
Even when using a pipette perfectly, errors can still occur. Therefore, it is recommended to perform at least 2, preferably 3 repetitions for each sample. This way, if a single well shows abnormal conditions, you can remove the outlier data and ensure high-quality results. When there are less than 3 repetitions, it can be difficult to determine which well is abnormal. Although performing more repetitions can consume more reagents, repetition is crucial to obtaining accurate data. While it may require multiple 96-well plates to analyze all samples, it will ultimately save time and costs by avoiding retesting.
04 Template for qPCR
Low-level amplification can result in high CT values, but due to the random nature of PCR amplification, low product levels can also lead to increased errors. In other words, the fewer templates, the greater the error in amplification because the reaction becomes more sensitive to changes in cycle number and template concentration.
To avoid errors from affecting the data, it is recommended to analyze the data before 30 cycles. After 30 cycles, the accuracy of the data cannot be guaranteed regardless of how skilled you are in pipetting. For samples with particularly low levels, increasing the cDNA concentration is the most effective solution. Generally, a 50% increase in concentration is enough to increase the CT value by 1-2.
05 Maintenance of Pipettes
Regular calibration of pipettes used for qPCR is essential for accuracy. The frequency of calibration should depend on the frequency of use, with a recommended interval of every 6-12 months. If you only perform relative quantification and the control group already includes housekeeping genes, the error may be constant and will not deviate significantly from the experimental results. However, when absolute quantification is required, uncalibrated pipettes can lead to serious bias in the data.
06 Buying a New Pipette
For qPCR reactions, multi-channel continuous pipettes are recommended to simplify the process. While in the past, multi-channel pipettes were associated with lower accuracy, with technological improvements, they can now offer higher accuracy than single-channel pipettes. A continuous pipette can be used to add Master Mix, dilute cDNA into an 8-tube strip, and then a multi-channel pipette can be used to add it to the 96-well plate.
07 Understanding the Composition of Reagents
When working with SYBR Green and other qPCR Master mixes, it's important to understand that these reagents typically contain glycerol and have a viscous consistency. If they are aspirated like water, it's likely that the aspirated volume will be too small. To avoid this issue, reverse pipetting can be used. While it does result in some wasted solution, this technique can pre-wet the inner wall of the pipette tip and reduce the error of pipetting, as well as prevent the formation of bubbles. This ensures more accurate and reliable results in your qPCR experiments.