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Difference between One-step RT-qPCR and Two-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...

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This article writter by MN Editors on May 27, 2021

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Difference between One-step RT-qPCR and Two-step RT-qPCR
Difference between One-step RT-qPCR and Two-step RT-qPCR

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

TopicOne-step RT-qPCRTwo-step RT-qPCR
DefinitionIn 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 RTOligo(dT) primers, Random hexamers, Gene-specific primers, A mix of theseGene-specific primers
Fresh RNA sampleNeed fresh RNA sample(s) to analyze new targets or repeat experimentsNo Need of fresh RNA sample.
ComplexcitySimple and fast analysisNot Simple and fast analysis test as compared to One-step RT-qPCR
Errors and contaminationLess chances of errors and contaminationThere are risk of errors and contamination
Pipetting stepLess pipetting stepsMore pipetting steps
FlexibilityDon’t provide flexibilityProvide Greater flexibility to select RT enzymes and DNA polymerases for PCR separately
Store cDNANoStore cDNA for later use
ApplicationFor high-throughput applicationsPreferred method for applications with limited amount of starting material (i.e. single cell analysis)
Timerequired a short period of time.required log period of time.
Open-tubeDoes notIt requires an extra open-tube step.
RNA sample reuseNoThe same RNA sample can be used for multiple targets due to separate reactions.
Closed-tube/Open-tubeClosed-tube reactions.Open-tube reactions.
HandlingMinimal sample handling, which reduced the bench time.Not
Best for1. 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
Considerations1. 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

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