qPCR goes further than endpoint PCR
Unlike endpoint reverse transcription PCR (RT-PCR)—used for determining the presence or absence of a particular gene product, real-time PCR or quantitative PCR (qPCR) can measure the starting copy number and detect small differences in expression levels between samples.
Don’t let these factors compromise reactions
Endpoint PCR is robust and almost always produces “a result”. However, ensuring that qPCR is quantitative, accurate, and reproducible requires thoughtful assay design, setup, and optimization. Poor qPCR assay design—imperfect reaction conditions (e.g., choice of master mix, primer, or probe Tm), presence of inhibitors, or underlying single-nucleotide polymorphisms (SNPs)—can lead to suboptimal amplification and extensive troubleshooting.
Validate chosen assays under your conditions
Use of previously identified assays (e.g., from the literature or an associated lab) that were not validated under your specific reaction conditions (e.g., RNA extraction method, assay reagents, thermal cycler), can result in having to repeat experiments. This wastes valuable time, reagents, and, most importantly, precious samples. Always test chosen assays using your experimental conditions, even if the assays were successfully used by someone else.
Consider the following recommendations
To achieve reliable, interpretable results from qPCR, the following important factors must be considered:
- Before beginning assay design, you should know your gene, its transcript variants, and their exon organization. Be aware of SNP positions so that you can avoid positioning primers and probes over them. You will also want to perform BLAST searches to ensure the primers and probe sequences you select are specific to the target.
- Primer and probe design are crucial to the success of the experiment. Melting temperature, length, and GC content all come into play. Aim for primer lengths of 18−30 bases, with the melting temperature of the 2 primers not differing by more than 4°C and the GC content falling within 35−65%. Probes are typically 20−30 bases long with melting temperatures 6−8°C higher than that for the primers.
- The real-time PCR instrument will dictate certain parameters of the experiment; importantly, some instruments are not compatible with certain fluorescent dyes. Be sure to refer to the instrument manufacturer’s guidelines to verify compatible dyes and correct cycling conditions. Also consider the use of special high-quenching probes that can minimize crosstalk between dye signals.
- If you are running a multiplex experiment, additional considerations will need to be incorporated, particularly in the assay design and choice of dyes. Each target must be identified by a separate reporter dye, with each having little to no overlap in emission spectra. That said, FAM is a particularly good dye to select for any low-copy transcript targets because it has a strong signal.
- As a final step before reaction setup, determine the controls that you will run. Include replicates as well as positive and negative controls. Typically, 3 technical and 3 biological replicates are run, along with an exogenous and endogenous positive control, and no-template, no-RT, and no-amplification negative controls. Be sure to calculate those extra reactions into reagent needs.
- Always use RNase- and DNase-free reagents, check their expiration dates, and verify their concentrations.
Learn the “why” for these recommendations
All of these points are examined in detail with best practice recommendations and rationale provided in the Real-time qPCR guide: Part 1—Assay design. This extensive application guide provides chapters on assay selection and design, use of replicates and controls, and choice of appropriate master mixes. Reporter dye selection and multiplex qPCR are also discussed. The guide is written by IDT scientists, and is free—simply register for it here.