One-step RT-qPCR
In One-step RT-qPCR or One-step real time Polymerase chain reaction (PCR) the reverse transcriptase and DNA polymerase are premixed into a single tube. This allows the RT step and subsequent amplification step to be performed in a single reaction.
Two-step RT-qPCR
In Two-step RT-qPCR or One-step real time Polymerase chain reaction (PCR) the reverse transcription of the RNA template is performed first. Once completed, the amplification of the cDNA is carried out in a separate reaction.
Difference between One-step RT-qPCR and Two-step RT-qPCR
| Topic | One-step RT-qPCR | Two-step RT-qPCR |
| Definition | In One-step RT-qPCR or One-step real time Polymerase chain reaction (PCR) the reverse transcriptase and DNA polymerase are premixed into a single tube. This allows the RT step and subsequent amplification step to be performed in a single reaction. | In Two-step RT-qPCR or One-step real time Polymerase chain reaction (PCR) the reverse transcription of the RNA template is performed first. Once completed, the amplification of the cDNA is carried out in a separate reaction. |
| Primers used in RT | Oligo(dT) primers, Random hexamers, Gene-specific primers, A mix of these | Gene-specific primers |
| Fresh RNA sample | Need fresh RNA sample(s) to analyze new targets or repeat experiments | No Need of fresh RNA sample. |
| Complexcity | Simple and fast analysis | Not Simple and fast analysis test as compared to One-step RT-qPCR |
| Errors and contamination | Less chances of errors and contamination | There are risk of errors and contamination |
| Pipetting step | Less pipetting steps | More pipetting steps |
| Flexibility | Don’t provide flexibility | Provide Greater flexibility to select RT enzymes and DNA polymerases for PCR separately |
| Store cDNA | No | Store cDNA for later use |
| Application | For high-throughput applications | Preferred method for applications with limited amount of starting material (i.e. single cell analysis) |
| Time | required a short period of time. | required log period of time. |
| Open-tube | Does not | It requires an extra open-tube step. |
| RNA sample reuse | No | The same RNA sample can be used for multiple targets due to separate reactions. |
| Closed-tube/Open-tube | Closed-tube reactions. | Open-tube reactions. |
| Handling | Minimal sample handling, which reduced the bench time. | Not |
| Best for | 1. Amplifying multiple targets from a single RNA source 2. When you plan to reuse cDNA for additional amplifications | 1. Working with multiple RNA samples to amplify only a few targets 2. Assays performed repeatedly |
| Considerations | 1. Requires more setup, hands-on, and machine time 2. Additional pipetting increases the chances for experimental errors and contamination 3. Uses more reagents | 1. Must “start over,” or save RNA aliquot and perform new RT to analyze new target or repeat amplifications 2. Reaction conditions are not optimal—efficiency & thus quantification are affected 3. Primer dimers a bigger potential problem |
References
- https://sg.idtdna.com/pages/education/decoded/article/one-step-two-step
- https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/choice-of-one-step-rt-qpcr-or-two-step-rt-qpcr
- https://www.thermofisher.com/in/en/home/brands/invitrogen/molecular-biology-technologies/spotlight-articles/onestep-vs-twostep-rtpcr.html
- https://microbiologynote.com/real-time-pcr-principle/#Standard_Protocol_of_One-Step_RT-qPCR
