Optimization Tips for Luna^® One-Step RT-qPCR

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 0.025 units/µl Antarctic Thermolabile UDG (NEB #M0372)  for 10 minutes at room temperature prior to thermocycling. Alternatively, use products already containing Antarctic Thermolabile UDG in the mix (e.g., NEB #M3019, L4001)
• A universal passive reference dye is included in the following Luna® qPCR products: Luna Universal qPCR Master Mix (NEB #M3003), Luna Universal Probe qPCR Master Mix (NEB #M3004), Luna Universal One-Step RT-qPCR Kit (NEB #E3005), and Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006). These products support broad instrument compatibility (High-ROX, Low-ROX, ROX-independent) so no additional ROX is required for normalization. For specific instances, No ROX mixes are available (NEB #E3007, M3029).
  
  The Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) contains no reference dye and is compatible with any instrument that does not require ROX. If ROX normalization is needed, ROX can be added. Please refer to instrument manufacturer’s instructions for details. 

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