PCR and qPCR Master Mixes

1/ Two buffers optimized for independent RT and real-time PCR 2/ Highly sensitive 3/ Recommended when the reaction is performed with a limiting amount of starting material 4/ More efficient because random primers and oligo d(T) can be used 5/ Possibility to stock cDNA to quantify several targets.

When performing a reverse transcription reaction for a two-step quantitative PCR assay, it is possible to use different primer strategies. Random hexamer primers, which bind anywhere on the RNA allow full coverage of total RNA (including ribosomal, bacterial and viral RNA), oligo d(T)n (or anchored oligo d(T)n), which binds to the poly-A tail of the mRNA which is located at the extremity of the transcript, leading to full transcripts, a mixture of random hexamer and oligo d(T)n that reduces the risk of bias in cDNA synthesis and specific primers, which bind to the transcript of interest. The ability to optimize both cDNA and quantitative PCR reactions separately and the higher cDNA yields, two-step reactions can be more sensitive than one-step reactions. The ability to separately optimize these reactions is also beneficial when performing multiplex qPCR on challenging sequences.

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 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.