qPCR: 1-Step vs 2-Step

Learn that gene expression varies across time, tissues, and conditions, and that RT-qPCR is a sensitive method for measuring RNA transcript abundance through reverse transcription followed by real-time amplification.

Generate cDNA from the RNA template using reverse transcriptase and random primers, oligo(dT), or a combination of both as the first step of two-step RT-qPCR.

Combine a portion of the synthesized cDNA with gene-specific primers and thermostable DNA polymerase to perform real-time amplification of the sequence of interest.

Perform reverse transcription and initial cDNA generation in a single reaction using reverse transcriptase and gene-specific primers on the RNA template.

Execute thermocycling where gene-specific primers and DNA polymerase amplify the sequence of interest within the same reaction tube.

Recognize that two-step RT-qPCR with oligo(dT) and random primers produces a cDNA pool that can be archived and used for multiple different qPCR assays on various target sequences.

Understand that one-step RT-qPCR offers convenience and reduces time and pipetting steps by combining both reactions in a single tube, though it sacrifices the ability to analyze multiple transcripts from one cDNA synthesis.