- Short PCR amplicons, ranging from 70 to 200 bp, are recommended for maximum PCR efficiency
- Target sequences should ideally have a GC content of 40–60%
- Avoid highly repetitive sequences when possible
- Use high quality, purified DNA templates whenever possible. Luna qPCR is compatible with DNA samples prepared through typical nucleic acid purification methods.
- Template dilutions should be freshly prepared in either TE or water for each qPCR experiment
- Generally, useful concentrations of standard and unknown material will be in the range of 106 copies to 1 copy. For gDNA samples from large genomes, (e.g., human, mouse) a range of 50–1 pg of gDNA is typical. For small genomes, adjust as necessary using 106–1 copy input as an approximate range. Note that for dilutions in the single-copy range, some samples will contain multiple copies and some will have none, as defined by the Poisson distribution.
- To generate cDNA, use of the ProtoScript® II First Stand cDNA Synthesis Kit (NEB #E6560) is recommended. Input 1 µg–0.1 pg of RNA as starting material.
- cDNA does not need to be purified before addition to the Luna reaction but should be diluted at least 1:10 before addition to qPCR
- Primers should typically be 15–30 nucleotides in length
- Ideal primer content is 40–60% GC
- Primer Tm should be approximately 60°C
- Primer Tm calculation should be determined with NEB’s Tm calculator using the Hot Start Taq setting
- For best results in qPCR, primer pairs should have Tm values that are within 3°C
- Avoid secondary structure (e.g., hairpins) within each primer and potential dimerization between primers
- G homopolymer repeats ≥ 4 should be avoided
- Optimal primer concentration for dye-based experiments (250 nM) is lower than for probe-based experiments (400 nM). If necessary, the primer concentration can be optimized between 100–500 nM for dye-based qPCR or 200–900 nM for probe-based experiments.
- Higher primer concentrations may increase secondary priming and create spurious amplification products
- When using primer design software, enter sufficient sequence around the area of interest to permit robust primer design and use search criteria that permit cross-reference against relevant sequence databases to avoid potential off-target amplification.
- For cDNA targets, it is advisable to design primers across known exon-exon junctions in order to prevent amplification from genomic DNA
- Primers designed to target intronic regions can ensure amplification exclusively from genomic DNA
- Probes should typically be 15–30 nucleotides in length to ensure sufficient quenching of the fluorophore
- The optimal probe concentration is 200 nM but may be optimized between 100 to 500 nM
- Both single or double-quenched probes may be used
- In general, non-fluorescence quenchers result in better signal-to-noise ratio than fluorescence quenchers
- Ideal probe content is 40–60% GC
- The probe Tm should be 5–10°C higher than the Tm of the primers to ensure all targeted sequences are saturated with probe prior to amplification by the primers
- Probes may be designed to anneal to either the sense or antisense strand
- Generally, probes should be designed to anneal in close proximity to either the forward or reverse primer without overlapping
- Avoid a 5´-G base which is known to quench 5´-fluorophores
- Avoid primer/probe combinations that contain complementary sequences, and ensure target sequences do not overlap
- Probes should be designed such that each amplicon has a unique fluorophore for detection
- Select fluorophores based on the detection capabilities of the available real-time PCR instrument
- The emission spectra of the reporter fluorophores should not overlap
- Test each primer/probe combination in a singleplex reaction to establish a performance baseline. Ensure Cq values are similar when conducting the multiplex qPCR.
- Pair dim fluorescence dyes with high abundance targets and bright dyes with low abundance targets
- Optimization may require lower primer/probe concentrations to be used for high copy targets along with higher concentrations for low copy targets
- Generally, best performance is achieved using the cycling conditions provided in the manual
- Longer amplicons (> 400 bp) can be used but may require optimization of extension times
- Due to the hot start nature of the polymerase, it is not necessary to preheat the thermocycler prior to use
- Select the “Fast” ramp speed where applicable (e.g., Applied Biosystems QuantStudio®)
- Amplification for 40 cycles is sufficient for most applications, but for very low input samples 45 cycles may be used
- For best results, keep reactions on ice prior to thermocycling
- A reaction volume of 20 µl is recommended for 96-well plates while a reaction volume of 10 µl is recommended for 384-well plates
- Reactions should be carried out in triplicate for each sample
- For each amplicon, ensure to include no template controls (NTC)
- To prevent carry-over contamination, treat reactions with 0.2 units/µl Antarctic Thermolabile UDG (NEB #M0372) for 10 minutes at room temperature prior to thermocycling
- The Luna reference dye supports broad instrument compatibility (High-ROX, Low-ROX, ROX-independent) so no additional ROX is required for normalization
- Ensure 90–110% PCR efficiency for the assay over at least three log10 dilutions of template
- Linearity over the dynamic range (R2) should ideally be ≥ 0.99
- Target specificity should be confirmed by product size, sequencing or melt-curve analysis
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