AAT Bioquest

When should I use one-step as opposed to two-step qRT-PCR?

Posted August 14, 2023


Consider these factors when deciding between one-step and two-step qRT-PCR:

One-step qRT-PCR combines first-step cDNA synthesis and subsequent PCR in a single reaction tube. This simplifies workflow, minimizes potential contamination, and reduces variation. It is suitable for processing large sample numbers, making it ideal for high-throughput applications.

On the other hand, two-step qRT-PCR involves separate reactions for first-strand cDNA synthesis and PCR, allowing the detection of multiple genes in a single RNA sample. This approach ensures specificity and efficiency.

Both methods have their advantages and disadvantages, so choose based on your specific experimental needs. One-step is efficient for quantifying the same gene(s) repeatedly, especially in high-throughput settings. It is also quicker, less expensive and involves less sample handling. The advantage of two-step is the flexibility it offers for detecting multiple genes in the same RNA sample.

Additional resources

Analysis of One-Step and Two-Step Real-Time RT-PCR Using SuperScript III

Real-Time PCR (qPCR)

Helixyte™ Green *20X Aqueous PCR Solution*

Tide Quencher™ 1 acid [TQ1 acid]