In one-step qPCR or reverse transcription qPCR, cDNA synthesis and qPCR are performed in a single reaction tube. In two-step qPCR, cDNA is synthesized in one reaction tube, and aliquots of the cDNA is then used for a subsequent RT-qPCR experiments.

• Accurate representation of target copy number
• Best option for high-throughput screening
• Simple and rapid
• Fewer pipetting steps (reducing possible errors and contamination)
• Best method when only a few assays are run repeatedly
• Multiplex qPCR of gene of interest and control can be done in single well, from same RNA sample

There are several other advantages of one-step reactions, these include limited sample handling and reduced bench time, which helps to decrease chances for pipetting errors and cross contamination between reverse transcription and qPCR steps. This method is quick to set up and makes processing multiple RNA samples easy (especially when using liquid handling robotics), when you are amplifying only a few genes of interest. It is therefore ideal for high throughput screening laboratories where only a few reverse transcription qPCR assays are run repeatedly, using well-established reaction conditions, with the added advantage that multiplex qPCR of the gene of interest and control genes can be done in single well, from same RNA sample.

Gene-specific primers are required for generating the cDNA and for subsequent amplification in one tube, however this reduces experimental variation, since both enzymatic reactions take place under the same conditions, making one-step qPCR highly reproducible.
One-step RT-PCR requires careful evaluation to prevent primer dimer formation, because the primers will be present during the lower temperature conditions of the RT reaction as well as the qPCR cycling. This is particularly important in multiplex qPCR assays.

There are several ways to avoid genomic contamination with reverse transcription qPCR. Firstly, design forward and reverse primers in exons upstream and downstream of a large intron. This strategy favors the smaller amplicon and will prevent the genomic DNA from being amplified.
Secondly, for genes that lack introns or when the genomic structure is unknown, pre-treat the RNA with DNase I to remove genomic DNA.

No, intercalating dyes bind into all DNA, this means that they cannot be used for multiplex qPCR, as different amplicons cannot be distinguished, whereas different dyes can be used on probes for different amplicons. This also makes probe-based systems more specific; only detect the gene of interest, even making it possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimization, and they can be used for multiplex qPCR.

When template RNA has stable secondary structures and when doing multiplex qPCR, it can be beneficial to extend the reaction time up to 20 minutes and/or the temperature use elevated temperatures (up to 48°C) in the reverse transcription reaction. If the RNA contains a lot of stable secondary structures (often seen in RNA viruses), we recommend using an RT-qPCR mix with a thermostable reverse transcriptase, such as Inhibitor-Tolerant RT-qPCR Mix (MDX016), where the reverse transcription reaction time can be extended up to 55° C.