Slope of standard 10-fold dilution curve indicates qPCR efficiency. The efficiency of the qPCR should be between 90–110% (−3.58 ≥ slope ≥ −3.1). If the efficiency is greater than 100%, this indicated inaccurate sample and reagent pipetting, if it is less than 100%, this indicates your samples may contain PCR inhibitors or your qPCR primer and/or probe design may not be optimal.

One-step reaction
• Accurate representation of target copy number
• Simple and rapid
• Fewer pipetting steps (reducing possible errors and contamination)
• Best option for high-throughput screening
• 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
Two-step reaction
• Two buffers optimized for independent RT and qPCR
• Highly sensitive
• Potentially more efficient because random primers and oligo d(T) can be used
• Possibility to stock cDNA to quantify several targets
• 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.

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.

Many fluorescent qPCR primer- and probe-based chemistries have been devised and are available from different commercial vendors for molecular diagnostic tests, the two most commonly used are:
• Hydrolysis (TaqMan) probes. The main advantages of using hydrolysis probes are high specificity, a high signal-to-noise ratio, and the ability to perform multiplex qPCR reactions. The disadvantages are that the initial cost of the probe.
• Molecular beacons. Molecular beacons are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, hybridization and fluorescence will not occur. Unlike hydrolysis probes, molecular beacons are displaced but not destroyed during amplification and can be used for melt curve analysis if necessary. The main disadvantage is that they are difficult to design, requiring a stable hairpin stem that is strong enough that the molecule will not spontaneously fold into non-hairpin conformations but not be too strong, or it may not properly hybridize to the target.

The Lyo-Ready qPCR and RT-qPCR master mixes have some inhibitor tolerance, but for amplification from crude lysates or inhibitor-rich samples we have the universal Inhibitor-Tolerant qPCR/RT-qPCR mixes and for even greater sensitivity, we have the sample specific (blood, saliva, urine, stool, and plant) mixes.

No-RT control reactions are useful for determining issues that may arise from the amplification of genomic DNA that may be present in a sample. However, many RNA transcripts are present at low abundance and for these transcripts, it is recommended to perform a No-RT control reaction if primer sets do not span exon-exon junctions. If the primers do span exon-exon junctions, or genomic DNA is not likely to interfere (when looking at RNA viruses in a sample for example) a No-RT control is not required.

Following lyophilization in the presence or absence of primers and probes, the Lyo‑Ready^™ qPCR and RT-qPCR master mixes 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.