One-step and Two-Step RT-qPCR
RT-qPCR has been a popular technique for gene expression analysis for recent years. We pretty sure that you have heard of one-step and two-step RT-qPCR, but did you know the difference between them? If no, we would explain in details below.
What is One-step RT-qPCR?
In one-step RT-qPCR, both the reverse transcription of RNA and qPCR take places in the same reaction tube. Addition of one-step PCR kit with designed primers into freshly prepared RNA sample is sufficient for the whole reaction to proceed. No additional tube opening for aliquoting and addition of reagents are necessary. This helps to minimize the possibility of pipetting error and cross-contamination that affect the efficacy of RT-qPCR.
The minimal sample handling throughout the whole reaction also makes one-step qPCR time-saving. However, the quality and scarcity of the RNA sample must be taken into consideration. This is because they are important in ensuring the reproducibility and reliability of the outcome of the qPCR reaction. In addition, due to the inability of one-step qPCR to aliquot and store the cDNA synthesized during reverse transcription, one-step qPCR requires a freshly prepared RNA sample to start with each reaction.
What is Two-step RT-qPCR?
In the case of two-step RT-qPCR, it involves two reactions to occur in different reaction tubes, in contrast to one-step. It includes reverse transcription and qPCR.
After RNA is reverse transcribed to cDNA, the cDNA need to be transferred into another reaction tube. The qPCR reagent is then added into the second tube to perform qPCR. Since two-steps RT-qPCR involves additional pipetting and transferring of cDNA, this method has a higher risk of contamination when compared to one-step RT-qPCR. As a result, pipetting and aseptic technique must be carried out carefully. This is to maximize the accuracy and reliability of the final outcome of qPCR.
Unlike one-step, in two-step RT-qPCR, the two reactions can be optimized separately. You can optimize by choosing different sets of reverse transcriptase and qPCR reagents corresponding to your RNA isolate of interest. This is particularly important for some difficult RT-qPCR reactions as you could get better qPCR results. Due to only a small amount of cDNA is required for proceeding with PCR, the synthesized cDNA during reverse transcription can be collected and stored for later use in future reactions.
Comparisons of One-step and Two-step RT-qPCR
| Description | RT Primers | Advantages | Disadvantages | Ideal Uses |
| One-step RT-qPCR | -Gene-specific primers | -Quick setup and limited hands-on time -Single closed-tube reaction, reducing contamination | -Need fresh RNA sample(s) to analyze new targets or repeat experiments | -Assessing many RNA samples -High-throughput applications |
| Two-step RT-qPCR | -Oligo(dT) primers -Random hexamer primers -Gene-specific primers -A combination of the above | -Choice of RT primers -Flexible reaction optimization (RNA input, choice of the enzyme(s), enzyme amount, & reaction | -More setup and hands-on time -Greater variation and risk of contamination due to extra open-tube step and pipetting | -Assessing multiple targets from a few RNA samples -Saving cDNA product for future re-use |
Luna®️ from New England Biolabs
Luna®️ products from New England Biolabs are optimized for both qPCR and RT-qPCR, and are available for both one and two-step RT-qPCR. You may visit Luna®️ qPCR and RT-qPCR series for more information.
