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.

• Two buffers optimized for independent RT and real-time PCR
• Highly sensitive
• Recommended when the reaction is performed with a limiting amount of starting material
• More efficient because random primers and oligo d(T) can be used
• 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 two-step quantitative PCR, the use of several tubes means that it is more time consuming and less adaptable to liquid handling robotics and so more difficult to adopt for high throughput
screening assays. The use of several tubes and pipetting steps also exposes the reaction to a greater risk of DNA contamination.

Many fluorescent qPCR primer- and probe-based chemistries have been devised and are available from different commercial vendors, including:
• Hydrolysis (TaqMan) probes
• Molecular beacons
• Dual hybridization probes
• Eclipse probes
• Amplifluor assays
• Scorpions PCR primers
• LUX PCR primers
• QZyme PCR primers

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.

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.