The Choice of RT-qPCR: One-step or Two-step
As a newbie who has just entered the laboratory , my senior in the laboratory recently purchased a one-step quantitative reagent, but I have been using the "reverse transcription followed by quantification" method. So what is the one-step quantitative reagent?
In order to keep up with my senior brother and not lag behind the laboratory, I studied and summarized the following materials. Real-time quantitative PCR (qRT-PCR or qPCR) is an important method for quantifying genes and gene transcription levels (i.e. RNA). For RNA quantification, two processes are required: reverse transcription (RT) and quantification (qPCR). According to the different experimental operation steps, it can be divided into: One-step RT-qPCR and Two-step RT-qPCR.
In order to keep up with my senior brother and not lag behind the laboratory, I studied and summarized the following materials. Real-time quantitative PCR (qRT-PCR or qPCR) is an important method for quantifying genes and gene transcription levels (i.e. RNA). For RNA quantification, two processes are required: reverse transcription (RT) and quantification (qPCR). According to the different experimental operation steps, it can be divided into: One-step RT-qPCR and Two-step RT-qPCR.
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One-step RT-qPCR
Experimental process: RT and qPCR are in the same reaction tube, which can achieve direct quantification of RNA.
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Advantages of the experimental method: 1. Multi-sample gene quantification: Based on the premix on the market, a Mix containing upstream and downstream primers of the gene to be tested can be prepared to detect the same gene in different samples to be tested. 2. Low probability of contamination: Fewer sample additions effectively reduce the probability of component contamination during system preparation; 3. Easy to operate and save time.
Considering that both RT and qPCR are enzymatic reactions, the core enzymes are Reverse Transcriptase and DNA Polymerase. If the two enzymatic reactions are carried out in the same system, the performance of one of them will inevitably be sacrificed. Therefore, this method has the following disadvantages: 1. RT process: ① High requirements for template RNA quality: The quality of RNA seriously affects the efficiency of reverse transcription; ② Large amount of template used: One-step qRT-PCR uses gene-specific primers, while the RT process of two-step qRT-PCR uses the optimal ratio of Oligo dT and Random Primer as primers. In terms of the amount of reverse transcription products, gene-specific primers are lower than Oligo dT and Random Primer; ③ It is easy to cause the formation of primer dimers: Under 37-50℃, the 3' ends of the upstream and downstream primers are prone to annealing, while the optimal enzyme activity temperature of Reverse Transcriptase is just 37-50℃. 2. qPCR process: ① If the quantitative method is dye method, primer dimers will interfere with the quantitative results; ② DNA polymerase activity is inhibited by certain components in the reverse transcription system, affecting the amplification efficiency.
The scientists have modified the structure of RT Enzymes and DNA Polymerase, optimized the enzyme reaction ion environment, reduced inhibitory components, and achieved sufficient efficiency for RT and qPCR reactions to be performed in the same system.
Considering that both RT and qPCR are enzymatic reactions, the core enzymes are Reverse Transcriptase and DNA Polymerase. If the two enzymatic reactions are carried out in the same system, the performance of one of them will inevitably be sacrificed. Therefore, this method has the following disadvantages: 1. RT process: ① High requirements for template RNA quality: The quality of RNA seriously affects the efficiency of reverse transcription; ② Large amount of template used: One-step qRT-PCR uses gene-specific primers, while the RT process of two-step qRT-PCR uses the optimal ratio of Oligo dT and Random Primer as primers. In terms of the amount of reverse transcription products, gene-specific primers are lower than Oligo dT and Random Primer; ③ It is easy to cause the formation of primer dimers: Under 37-50℃, the 3' ends of the upstream and downstream primers are prone to annealing, while the optimal enzyme activity temperature of Reverse Transcriptase is just 37-50℃. 2. qPCR process: ① If the quantitative method is dye method, primer dimers will interfere with the quantitative results; ② DNA polymerase activity is inhibited by certain components in the reverse transcription system, affecting the amplification efficiency.
The scientists have modified the structure of RT Enzymes and DNA Polymerase, optimized the enzyme reaction ion environment, reduced inhibitory components, and achieved sufficient efficiency for RT and qPCR reactions to be performed in the same system.
Two-step RT-qPCR
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For most students, this is the most basic and classic experimental technique they learn when they enter the laboratory. The experimental process is: RT first, then qPCR, and indirectly quantify RNA by quantifying cDNA.
Advantages of the experimental method: RT process: 1. Broad RNA requirements: Most RT Enzymes can use 1ng-1μg of total RNA as the starting template for high-efficiency reverse transcription (the laboratory often buys HifairTM RT Enzymes from Xiaoyi, which is compatible with 1pg-5μg of total RNA as the starting template); 2. Wide application: cDNA can be used for library construction or gene cloning, etc. qPCR process: 1. High amplification efficiency: cDNA produced by the RT process can be diluted 5-20 times to minimize the inhibition of quantitative reactions; 2. High-throughput multi-gene quantification: cDNA samples obtained by reverse transcription can simultaneously detect multiple genes.
This method separates the RT and qPCR enzymatic reactions, which is more convenient for the release of RT Enzymes and DNA Polymerase enzyme activities. However, the step-by-step operation will take a long time and require multiple sample additions and pipetting, which may increase the chance of errors and cross-contamination.
Advantages of the experimental method: RT process: 1. Broad RNA requirements: Most RT Enzymes can use 1ng-1μg of total RNA as the starting template for high-efficiency reverse transcription (the laboratory often buys HifairTM RT Enzymes from Xiaoyi, which is compatible with 1pg-5μg of total RNA as the starting template); 2. Wide application: cDNA can be used for library construction or gene cloning, etc. qPCR process: 1. High amplification efficiency: cDNA produced by the RT process can be diluted 5-20 times to minimize the inhibition of quantitative reactions; 2. High-throughput multi-gene quantification: cDNA samples obtained by reverse transcription can simultaneously detect multiple genes.
This method separates the RT and qPCR enzymatic reactions, which is more convenient for the release of RT Enzymes and DNA Polymerase enzyme activities. However, the step-by-step operation will take a long time and require multiple sample additions and pipetting, which may increase the chance of errors and cross-contamination.
One-Step or Two-Step?
Here is the compiled characteristics and applications of the two methods so that we can better choose the quantitative method in subsequent experiments.
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