One-step reaction 1/ Accurate representation of target copy number 2/ Simple and rapid 3/ Fewer pipetting steps (reducing possible errors and contamination) 4/ Best option for high-throughput screening 5/ Best method when only a few assays are run repeatedly 6/ Multiplex qPCR of gene of interest and control can be done in single well, from same RNA sample. Two-step reaction 1/ Two buffers optimized for independent RT and qPCR 2/ Highly sensitive 3/ Potentially more efficient because random primers and oligo d(T) can be used 4/ Possibility to stock cDNA to quantify several targets 5/ Recommended when the reaction is performed with a limiting amount of starting material.

So, one-step workflows are commonly favoured in molecular diagnostic tests. Two-step RT-qPCR is preferred when multiple interrogations will be made of the same starting material or where archiving of cDNA may be required.

With a chemical hot-start DNA polymerase, the polymerase is covalently linked with chemical groups to block enzyme activity at room temperature. Advantages 1/ More stringent than antibody hot-start methods, so there is no activity even for long periods at room temperature, minimizes non-specific amplification 2/ Low bioburden, as it is free of animal and bacterial-origin components Considerations 3/ Longer activation time required for the polymerase to become fully active.

Aptamers are engineered oligonucleotides that bind to the active site of the high-fidelity DNA polymerase but are denatured and released as the temperature is increased during the PCR amplification cycle. This aptamer-mediated inhibition/activation process is however fully reversible, and so at the end of thermal cycling, when the temperature of the reaction is decreased, the aptamer refolds and rebinds to high-fidelity DNA polymerase, inhibiting any further activity in the sample. This has proven to be important in workflows where undesired polymerase activity after reaction completion can disturb baseline readings, such as NGS following amplification of a library.

Yes, reaction conditions are the same and they will produce the same Ct values, even with multiplex RT-qPCR assays. This means that the liquid mixes can be used to create SOPs if you do not want to lyophilize and then later if lyophilization is required (for example to increase sensitivity), the SOP can be updated for lyophilization, it does not require completely new SOPs to be written.

Following drying in the presence or absence of primers and probes, they are stable for a minimum of 24 months at ambient temperature (17 – 23 °C). Following rehydration, the assay reproducibility, sensitivity, and robustness will be the same as for a freshly made liquid mix, making the mixes ideal for point-of-care molecular diagnostic tests and microfluidic qPCR devices.