Target Selection

• 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
• Target sequences containing significant secondary structure should be avoided

RNA Template

• Use high quality, purified RNA templates when­ever possible. Luna qPCR is compatible with RNA samples prepared through typical nucleic acid purification methods.
• Prepared RNA should be stored in an EDTA-containing buffer (e.g., 1X TE) for long-term stability
• Template dilutions should be freshly prepared in either TE or water for each qPCR experiment
• Treatment of RNA samples with DNase I (NEB #M0303) may minimize amplification from genomic DNA contamination
• Generally, useful concentrations of standard and unknown material will be in the range of 10⁸ copies to 10 copies. 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. For total RNA, Luna One-Step Kits can provide linear quantitation over an 8-order input range of 1 µg–0.1 pg. For most targets, a standard input range of 100 ng–10 pg total RNA is recommended. For purified mRNA, input of = 100 ng is recommended. For in vitro-transcribed RNA, input of ≤ 10⁹ copies is recommended.

Primers

• 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
• The optimal primer concentration for dye-based experiments and probe-based experiments is 400 nM. If necessary, the primer concentration can be optimized between 100–900 nM.
• 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
• It is advisable to design primers across known exon-exon junctions in order to prevent amplification from genomic DNA

Hydrolysis Probes

• 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

Multiplexing

• 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 C_q 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

Reverse Transcription

• The default reverse transcription temperature is 55°C
• For difficult targets, the temperature of reverse transcription may be increased to 60°C for 10 minutes
• Due to the WarmStart feature of the Luna RT, reverse transcription temperatures lower than 50°C are not recommended

Cycling Conditions

• 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 dual WarmStart/Hot Start feature of the Luna kits, 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

Reaction Setup

• 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)
• A no Luna RT control should be conducted to guarantee amplification is specific for RNA input and not due to genomic DNA contamination
• 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

Assay Performance

• 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