When designing an RT-qPCR experiment, one of the first decisions is whether to use a one-step or two-step workflow. This guide compares one-step vs. two-step RT-qPCR, outlines key workflow differences, and provides reagent options to help you choose the most suitable RT-qPCR approach for your RNA detection application.

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Working with RNA samples

Since real-time PCR (qPCR) cannot be performed directly on RNA, RNA samples must first be copied to complementary DNA (cDNA) in a process called reverse transcription (RT). After reverse transcription, the cDNA can then be amplified using qPCR. 

Applications that start with RNA samples include gene expression analysis detecting messenger RNA (mRNA) and virus detection looking at viral RNA. Both applications offer either a one-step or two-step qPCR workflow, each with its own benefits and drawbacks.

DNA samples do not require the reverse transcription step and can be directly amplified by qPCR.

How 1-step and 2-step RT-qPCR workflows differ

One-step RT-qPCR workflow

A one-step master mix enables both reverse transcription and qPCR to be performed in one tube. It includes everything required to reverse transcribe RNA to cDNA and then amplify DNA by qPCR. There is no need to open the tube to add additional reagents.

The one-step reaction has fewer steps, less pipetting, and a lower risk of cross-contamination. It can be automated using robotics and thus allows for high-throughput sample processing. One-step is exceptional for testing many samples with one to a few target sequences. One drawback is that the cDNA cannot be stored since it is part of the one-step reaction. A two-step workflow should be used if you need to use your cDNA to analyze many targets.

Two-step RT-qPCR workflow

A two-step workflow involves performing reverse transcription first, to synthesize cDNA. Once completed, a small portion of the cDNA is transferred to a separate tube along with qPCR master mix and a target-specific assay. qPCR amplification of cDNA is then performed.

The cDNA generated during the reverse transcription step can be stored for further testing. Two-step is an excellent choice when customers need to screen many target sequences in each sample. It does, however, involve more steps and more pipetting.

The cDNA generated during the reverse transcription step can be stored for further testing. Two-step is the better choice when customers need to screen many target sequences in each sample. It does, however, involve more steps and more pipetting.

When should you use 1-step vs. 2-step RT-qPCR?

 One-step workflowTwo-step workflow
Features
  • Master mix contains both RT and qPCR reagents
  • Reduces chance of cross-contamination
  • Faster time-to-results
  • Easy for high-throughput workflow
  • RT is purchased separately from master mix
  • cDNA can be stored for further experiments
Use caseUse when testing many samples with one or few targetsUse when testing fewer samples with many targets
Primary considerationcDNA cannot be storedMultiple steps, slower to obtain results

Featured RT-qPCR master mixes

One-step master mixesPerformance summary
Power SYBR Green RNA-to-CT 1-Step Kit
SYBR Green–based one-step RT-qPCR kit enabling RNA-to-CT detection in a single tube, supporting gene expression analysis with minimal handling steps.
TaqMan Fast Virus 1-Step Master MixOptimized for rapid, sensitive one-step RT-qPCR, with performance maintained in the presence of common PCR inhibitors. Available with Rox and No Rox.
TaqPath One-step master mixesPerformance summary
TaqPath 1-Step Multiplex Master MixDesigned for multiplex RT-qPCR, enabling detection of up to four targets in a single reaction. Available in Mustang Purple and No Rox formats.
TaqPath DuraPlex 1-Step RT-qPCR Master MixMultiplex-optimized one-step RT-qPCR master mix designed for durable amplification performance and consistent target detection across multi-target RNA workflows. Available with Rox and No Rox.

Learn more about 1-step rt-qPCR master mixes >

Two-step master mixesPerformance summary
TaqMan Fast Advanced Master MixFormulated for broad dynamic range and reliable quantification in singleplex and duplex real-time PCR assays, including challenging targets.
PowerTrack SYBR Green Master MixSYBR Green master mix with tracking dye to support pipetting accuracy, single-copy detection, and broad instrument compatibility.
PowerUp SYBR Green Master MixDual hot-start SYBR Green master mix supporting high specificity, single-copy detection, and consistent performance across instruments.
TaqPath Two-step master mixesPerformance summary

TaqPath ProAMP Master Mix

TaqPath ProAMP Multiplex Master Mix

High-throughput master mixes for genotyping and copy number variation analysis, supporting specificity and reproducibility in the presence of inhibitors.
TaqPath qPCR Master Mix, CG2X master mix for gene expression and miRNA analysis, containing a thermostable DNA polymerase in an optimized buffer system for consistent amplification.
TaqPath BactoPure Microbial Detection Master Mix2X formulation for low-level microbial detection, supporting multiplex configurations (ROX for up to 3 targets; No ROX for higher-order multiplexing).

Understanding reverse transcription workflow options in RT-qPCR

For Research Use Only. Not for use in diagnostic procedures.