Thermo Fisher StepOne™ Real-Time PCR System

Overview

The Thermo Fisher StepOne™ Real-Time PCR System is a compact, benchtop real-time quantitative polymerase chain reaction (qPCR) instrument engineered for precision, reproducibility, and operational simplicity in molecular biology laboratories. It employs solid-state LED excitation and high-sensitivity photodiode detection to enable accurate, multiplexed fluorescence monitoring during thermal cycling. The system operates on the principle of sequence-specific amplification coupled with real-time fluorescence signal acquisition—enabling quantification of nucleic acid targets via threshold cycle (Ct) analysis, melting curve profiling, and relative or absolute quantitation. Designed for both novice users and experienced researchers, the StepOne™ system delivers validated performance for low-to-moderate throughput applications without requiring dedicated infrastructure or complex calibration routines. Its integrated 48-well aluminum thermal block ensures rapid heat transfer and stable temperature control, while the pre-aligned optical path eliminates routine alignment maintenance.

Key Features

• Compact footprint (29 × 33 × 35 cm) optimized for space-constrained labs, including core facilities and teaching environments.
• Three-channel optical detection system with factory-calibrated LEDs and bandpass filters for FAM™/SYBR® Green, VIC®/JOE™, and ROX™ dyes—enabling duplex and triplex assays without spectral overlap correction.
• Integrated 7-inch resistive touchscreen LCD with USB host port, supporting standalone operation without external PC connection for setup, run initiation, and basic result review.
• Thermal block accuracy of ±0.25 °C at 95 °C and uniformity of ±0.5 °C across all 48 wells—meeting ASTM E1532-16 requirements for thermal performance validation in qPCR instrumentation.
• Onboard StepOne™ Software v2.3+ with full instrument control, real-time data visualization, Ct calling, melt curve analysis, and HRM (High-Resolution Melting) support.
• Built-in RNAse P verification plate and calibration standards included for daily performance verification per ISO/IEC 17025 laboratory quality management guidelines.

Sample Compatibility & Compliance

The StepOne™ system accepts standard 0.2 mL thin-wall PCR tubes and 48-well optical reaction plates compatible with TaqMan® Assays, SYBR® Green I master mixes, and other commercially available qPCR reagents. All consumables are certified DNase/RNase-free and PCR-grade. The instrument and software comply with essential regulatory frameworks for life science research: data export in RDML format satisfies Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines; audit-trail functionality (enabled by default) supports GLP and GMP-aligned workflows; and electronic records meet FDA 21 CFR Part 11 requirements when deployed with appropriate IT controls and user access management.

Software & Data Management

StepOne™ Software provides an intuitive graphical interface for experiment design, thermal profile configuration, and post-run analysis. Key capabilities include: automated baseline and threshold setting; multiplate comparison tools; ΔΔCt and Pfaffl-based gene expression calculations; SNP genotyping clustering algorithms; and customizable report generation. Data can be exported in CSV, Excel, or MIQE-compliant RDML formats. Remote monitoring is supported via local network access—users receive email notifications upon run completion and may view real-time amplification plots from any authorized workstation. Primer Express® software is bundled for probe and primer design, ensuring compatibility with TaqMan® chemistry and minimizing assay optimization time.

Applications

The StepOne™ system is routinely deployed in academic, clinical research, and biopharmaceutical settings for: gene expression profiling (including low-abundance transcripts), SNP genotyping and allelic discrimination, microRNA quantification using stem-loop RT-qPCR, viral load determination (e.g., HIV, HCV, SARS-CoV-2), fusion transcript detection, copy number variation (CNV) analysis, and pathogen identification. Its validated compatibility with Life Technologies’ portfolio of over 3 million pre-qualified TaqMan® Assays enables immediate deployment for hundreds of human, mouse, rat, and model organism targets—reducing assay development timelines and improving inter-laboratory reproducibility.

FAQ

Is the StepOne™ system compatible with third-party reagents and probes?
Yes—the instrument accepts any qPCR chemistry that emits fluorescence within its three detection channels (450–550 nm, 550–600 nm, and 600–650 nm), including SYBR® Green I, EvaGreen®, and custom-labeled probes.
Can melt curve data be acquired and analyzed on the StepOne™ platform?
Yes—software versions 2.1 and later support high-resolution melt (HRM) acquisition with ramp rates as low as 0.025 °C/sec and data analysis for genotyping, methylation screening, and variant detection.
Does the system support remote instrument control or data access?
Remote monitoring is supported via HTTP-based local network access; however, full instrument control (e.g., program upload or parameter modification) requires direct USB or LAN connection to a Windows® host running StepOne™ Software.
What validation documentation is provided with the instrument?
Each unit ships with a Certificate of Conformance, RNAse P verification plate, and IQ/OQ documentation templates aligned with ISO/IEC 17025 and CLSI EP17-A2 standards.
Is software update support still available for legacy Windows® operating systems?
Thermo Fisher discontinued active software development for Windows® XP and Vista® after 2018; however, StepOne™ Software v2.3 remains fully functional on these platforms under extended security maintenance protocols.