New England Biolabs, E3005E, Luna® Universal One-Step RT-qPCR Kit

Related Categories Luna® qPCR & RT-qPCR,, PCR, qPCR & Amplification Technologies Applications Dye-based qPCR & RT-qPCR,, qPCR & RT-qPCR,, Two-Step RT-qPCR, FAQ Q: How do I use qPCR to determine the concentration of my material? A: Quantitative PCR (qPCR) uses real-time fluorescence to measure the quantity of DNA present at each cycle during a PCR. A wide variety of approaches have been developed for generating a fluorescent signal, the most common of which use either hydrolysis probes (e.g., TaqMan®), or a double-stranded DNA binding dye, (e.g., SYBR® Green). At a point where the qPCR fluorescence signal is detectable over the background fluorescence, a quantification cycle, or Cq value, can be determined. Cq values can be used to evaluate relative target abundance between two or more samples. Alternatively, they can be used to calculate absolute target quantities in reference to an appropriate standard curve, derived from a series of known DNA dilutions. Typically, qPCR instrument software will perform the necessary plotting and calculations for concentration determinations. Alternatively, the NEB online tools include a qPCR calculator, which can be found at NEBiocalculator.neb.com. Q: Can I set up my Luna® RT-qPCR at room temperature? A: Yes. Luna RT-qPCR can be set up at room temperature, but for best results, reactions should be kept on ice prior to thermocycling. Both enzymes present in the kit, Hot Start Taq DNA Polymerase and WarmStart® Luna Reverse Transcriptase, are controlled by temperature-sensitive reversible inhibitors (SOMAmer®). These SOMAmers inhibit polymerase activity below 45°C, preventing undesired nonspecific activity until the reaction is heated above the release temperature during the reverse transcription step. Q: What is the difference between probe- and dye-based versions of the Luna® qPCR Mixes? A: qPCR is typically measured in one of two ways: via an intercalating dye that fluoresces more strongly upon binding to double-stranded DNA, or via a fluorescently-labeled “probe” oligonucleotide that anneals to a specific sequence in the PCR amplicon. Background fluorescence of the probe is prevented by the presence of a quenching group. In the case of hydrolysis probes, the quencher is separated from the fluorophore during amplification by the 5´→3´ nuclease activity of Taq DNA Polymerase. To enable dye-based detection, the Luna Universal qPCR Mix contains an intercalating dye that is detected in the SYBR® Green or FAM channel of most real-time instruments. The Luna Universal Probe qPCR products do not contain this double-stranded DNA binding dye (since it would interfere with the use of FAM-labeled probes). Users must supplement the mix with a labeled probe oligonucleotide in addition to forward and reverse PCR primers. Please note that the passive reference dye present in both mixes does not interfere with the use of ROX labeled probes. Q: Should I use probe- or dye-based detection for my qPCR assays? A: Dye-based detection (e.g., SYBR® Green) requires only addition of PCR primers, making it a cost-effective qPCR option. However, the intercalating dye will detect any dsDNA produced in the reaction. Therefore, off-target and non-template amplification (NTC) can be observed for some primer sets, resulting in inaccurate quantification. Denaturation (melt) curves performed after the PCR can be used to distinguish between correct and nonspecific products. Additionally, only a single amplicon can be measured in a dye-based qPCR with no ability to perform multiplex reactions. Probe-based detection requires designing and obtaining a sequence-specific fluorescently-labeled probe oligonucleotide in addition to typical PCR primers. This increases assay costs, but probe-based qPCR experiments benefit from extreme specificity and are unlikely to result in inaccurate quantification due to NTC amplification. Multiplex reactions are possible with probes, as different amplicons can be designed with unique fluorophores according to the optical capabilities of the qPCR instrument. Q: How should I design primers for Luna® qPCR? A: We recommend using primer design software (e.g., Primer3) to select target and primer sequences in order to maximize amplification efficiency while minimizing nonspecific amplification and primer dimers. Luna qPCR is also compatible with commercially available qPCR assays. If designing primers manually, we encourage designing short amplicons (70 bp to 200 bp) with balanced GC content (40-60%). Aim for a Tm of approximately 60°C using Hot Start Taq settings in the NEB Tm calculator (TmCalculator.neb.com). For cDNA and RNA targets, it is advisable to design primers across known splicing sites (exon-exon junctions) in order to prevent amplification from genomic DNA. Q: How long should my amplicon be for qPCR? A: An important factor in a qPCR experiment is maximizing the efficiency of the PCR amplification, and short PCR amplicons help ensure high efficiency. Typical qPCR amplicons are 70-200 bp in length, and we recommend designing your target in that range. Longer amplicons can be used, but may require optimization (e.g., increased extension times). Q: Why is the Luna® qPCR Mix blue? Will this dye interfere with detection? A: The Luna qPCR Mix contains an inert visual reference dye that gives the mix a clear blue color. This colored appearance makes it easier to identify which wells of a qPCR plate have been loaded with reagents, which can be difficult with the small well size and opaque nature of many 96- and 384-well qPCR plates. The blue dye has been extensively evaluated in PCR and qPCR and it does not inhibit or interfere with qPCR detection. Q: Can I run the Luna® qPCR Mix on my qPCR instrument? A: The following Luna qPCR mixes contain a universal passive reference dye that enables compatibility with a wide variety of real-time instruments, including those that use no passive reference normalization dye (e.g. Bio-Rad® CFX), a low concentration passive reference (e.g. Applied Biosystems® 7500 or QuantStudio®), or a high concentration passive reference (e.g. Applied Biosystems 7900 or StepOne®). Luna Universal qPCR Master Mix (NEB #M3003) Luna Universal Probe qPCR Master Mix (NEB #M3004) Luna Universal One-Step RT-qPCR Kit (NEB #E3005) Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) The Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) and Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029) lack a reference dye and can be used with any instrument that does not require ROX normalization. Please refer to instrument manufacturer’s instructions for greater details. Additionally, the Luna Mix can be used with standard or fast cycling conditions. Q: Can I use fast instrument settings with the Luna® qPCR Mix? A: The Luna qPCR Mix is compatible with both fast and standard ramp speeds on instruments that offer both. We recommend specific cycling temperatures and times (see protocol or manual for more information) but either ramp setting can be used with these cycling conditions. (Note: cycling conditions should be verified after changing ramp speed setting; on certain real-time instruments, changing ramp speed will also revert cycling conditions to the instrument default setting.) Q: Do I need to add ROX? A: Some real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by machine limitations such as “edge effect”, bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. However, ROX normalization does little to the variations caused by pipetting errors of templates/primers, heterogeneous mixing, and evaporation/condensation issues. The following Luna® qPCR products contain a universal passive reference dye that enables compatibility with a variety of real-time instruments, including those that use no passive reference normalization and those that use a low or high amount of passive reference dye (ROX). Luna Universal qPCR Master Mix (NEB #M3003) Luna Universal Probe qPCR Master Mix (NEB #M3004) Luna Universal One-Step RT-qPCR Kit (NEB #E3005) Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) The Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) ) and Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029) lack a reference dye and can be used with any instrument that does not require ROX normalization. Please refer to instrument manufacturer’s instructions for greater details. qPCR Instrument ROX Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche Lightcycler® Qiagen Rotor-Gene™ Eppendorf Mastercycler® Cepheid® SmartCycler® Not recommended/required Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™ Agilent Mx™ Low ROX (~50 nM final) Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX (~500 nM final) Q: How many dilutions should I use to make a standard curve? A: Typical qPCR experiments include standards spanning 5 orders of magnitude (five 10-fold serial dilutions) to generate a standard curve. Choose a lowest standard amount that will represent approximately one copy of target when maximum sensitivity is desired. The number of points on the standard curve can be increased or decreased as desired, and as few as three points can be used when the target, assay, and sample are well characterized. Q: Why does NEB recommend 40-45 cycles? A: Amplification of 40 cycles is sufficient for high copy targets, or when using a well-characterized assay where non-template amplification is not a concern. We suggest 45 cycles to maximize confidence in detecting low- to single-copy targets. Additional cycles may also help detect non-specific amplification for a given primer set when using dye-based qPCR mixes. Q: Does the Luna® qPCR Mix contain dUTP? Can I use carryover contamination prevention methods? A: Yes, the Luna qPCR Mix is formulated with a mixture of dTTP and dUTP. This ensures both efficient PCR amplification as well as the incorporation of dU into the products during qPCR. qPCR products containing dU serve as a substrate for uracil DNA glycosylase, allowing carryover contamination prevention. If an amplicon is intended for routine use, if qPCR vessels will be opened, or if avoidance of carryover contamination is desired, 0.025 units/μL Antarctic Thermolabile UDG (NEB #M0372) can be added to the reaction mix. When including Antarctic Thermolabile UDG, set up the qPCR experiments at room temperature or include an additional 2 minute incubation step at 25°C before the default cycling conditions. Q: Why do I have multiple peaks in my melt curve? A: A typical denaturation (melt) curve performed after qPCR cycling with an intercalating dye, will typically give rise to a single distinct peak in the plot of the negative derivative of fluorescence vs. temperature. This indicates that the amplified double-stranded DNA products are a single discrete species. The presence of multiple DNA species in the same reaction can give rise to multiple peaks in the melt curve, typically indicating the presence of contaminating or off-target amplification products. Search the PCR primers against the input DNA sequence to determine if a product other than the desired target can be amplified, for example, due to sequence similarity or multiple forms of the same gene sequence. Redesign primers if necessary. Occasionally, due to sequence-specific characteristics and/or GC content, certain qPCR products will experience multi-stage melting transitions, giving rise to a melt curve with multiple peaks. These types of products can be treated as a successful qPCR. The melt profile of any sequence of interest can be predicted using the online tool uMelt. Q: How can I distinguish non-template amplification (NTC) from real products? A: The simplest way to tell if non-template amplification is occurring and affecting accurate quantitation is to include control reactions that contain the primers of interest but exclude the target input (with only sample buffer or water added in its place). These no-template control (NTC) reactions may produce amplification products, which will usually be of a different size and/or sequence content than the desired DNA product and will therefore denature with a different melt profile. Comparing melt curves from the different reactions can enable detection of any non-template amplification. Any reactions showing a melt profile similar to the NTC should be removed from the standard curve and quantitation calculations. Q: Why do I see amplification curves in my NTC samples? A: Products can appear in non-template control (NTC) wells due to the ability of some primer sequences to form structures that can be used as substrates by DNA polymerases. The most common version of this problem is the “primer dimer”, wherein some small regions of complementarity in the two qPCR primers permit the production of an amplifiable structure. Many primer design tools will aim to eliminate this possibility, and to ensure that no primer can form a secondary structure within its own sequence. This type of amplification can be discerned by evaluating melt curve profiles, as a primer dimer or nonspecific type amplification will result in DNA products with a melt peak different from that of the correct qPCR product. A second explanation for amplification in NTC wells is the presence of contaminating PCR products amplified in earlier qPCR experiments. This is a significant concern when the same assay is used repeatedly, and in particular when the reaction vessels are opened after the qPCR is complete. General sterile procedures can avoid this problem, such as not opening vessels after reactions or doing so in a satellite laboratory area with separate equipment and routine decontamination of laboratory space and equipment. Melt curves can be used to determine if the NTC amplification is caused by carryover contamination. If this is the case, the products in the NTC reactions will be the same species as the intended target and the melt profiles will match accordingly. If contamination is detected, then all reagents (primers, water, qPCR mix) should be replaced, equipment should be decontaminated with a 10% bleach solution, and sterile procedures should be used for future experiments. Use of 0.2 U/μL Antarctic Thermolabile UDG (NEB #M0372) can help mitigate carryover contamination for situations where it is a persistent problem or in particularly sensitive applications. Q: How much primer should I use with the Luna® Universal RT-qPCR Kit? A: For most targets, a final concentration of 400 nM for each primer will provide optimum performance. If needed, the final primer concentration can be optimized between 100–900 nM. Q: How do I choose between one-step RT-qPCR and two-step RT-qPCR? A: Two methods are available for quantifying RNA samples: one-step RT-qPCR and two-step RT-qPCR. In both cases, RNA is first reverse transcribed into cDNA, which is then used as the template for qPCR amplification. Reverse transcription can be performed separately from qPCR (i.e., two-step RT-qPCR) or directly in the qPCR mix (i.e., one-step RT-qPCR). One-step workflows are commonly favored in molecular diagnostic assays. Two-step RT-qPCR is preferred when multiple interrogations will be made of the same starting material or where archiving of cDNA may be required. For more details, please refer to “Choice of One-Step RT-qPCR or Two-Step RT-qPCR ”. Q: What RNA samples can be used in RT-qPCR with the Luna® Mix? A: For best results, we recommend an extraction or purification of the RNA starting material. The Luna RT-qPCR Kit works with nucleic acids from a variety of sample types purified using typical column-based methods (e.g. Monarch® RNA Miniprep Kit (NEB #T2010)). When performing studies using mammalian cell cultures, cell lysates generated by Luna Cell Ready Lysis Module (NEB #E3032) can be added to all Luna one-step RT-qPCR reagents. In addition, numerous studies have directly demonstrated the compatibility of Luna reagents with saliva and other clinically relevant samples using crude sample prep protocols. SalivaDirect is one example. Q: How much RNA template should I use in my RT-qPCR reaction? A: The Luna® Universal One-Step RT-qPCR Kit works well with purified RNA templates including total RNA, mRNA, and in vitro transcript samples. Amount in 20 μl reaction total RNA ≤ 1 μg poly(A)-RNA ≤ 0.1 μg in vitro transcripts ≤ 10⌃9 copies Q: Can I use longer targets in one-step RT-qPCR? A: In general, smaller amplicons (70 bp-200 bp) tend to result in higher efficiencies and earlier Cqs. For targets in this size range, a 30 second extension time is recommended. Longer targets will benefit from longer extension times but the actual time will depend on the length of the target and instrument type, and should be determined empirically. Q: What temperature should I use for cDNA synthesis with Luna® RT-qPCR kits? A: The thermostable Luna WarmStart® Reverse Transcriptase supports efficient cDNA synthesis at elevated temperatures compared to MMLV. In general, cDNA synthesis can be accomplished with a 10 minute incubation at 55°C. For difficult templates, the cDNA synthesis temperature can be increased to 60°C. Q: Should I include a no Luna® WarmStart® Enyzme Mix control (-RT Control)? A: Yes. Amplification in the absence of the reverse transcriptase in RT-qPCR generally indicates there is contaminating DNA in your RNA input. Treating your RNA samples with DNase will remove any DNA prior to performing RT-qPCR. Q: What is the fluorescent, double-stranded DNA binding dye in the Luna® qPCR master mix? A: The dye in the Luna qPCR master mix is a fluorescent intercalating dye with similar excitation and emission properties to SYBR®. The dye fluoresces with an emission maximum of ~535 nm when bound to double-stranded DNA (green circles) but displays weak fluorescence in the presence of single-stranded DNA or in the absence of DNA (orange triangles). To enable dye-based detection, select the SYBR Green or FAM channel of most real-time instruments. Q: Is the Monarch® Total RNA Miniprep Kit compatible with Luna® RT-qPCR Reagents? A: Yes. RNA purified with these kits can be used for RT-qPCR with all of NEB’s Luna One-Step RT-qPCR Kits and the LunaScript® RT products. RT-qPCR of Monarch-purified RNA purified from various sample types consistently produces high-quality results, allowing accurate and reproducible quantitation To demonstrate the quality and utility of Monarch Total RNA for RT-qPCR-based quantitation, 16 experiments were performed and visualized using the “dots in boxes” approach developed at NEB. Each experiment encompasses triplicate reactions for a five-log range of input template concentrations and no-template controls (NTC) (18 reactions total). A high-quality RT-qPCR experiment, represented by a dot inside the box, is defined as having an efficiency between 90-110%, a ΔCq of ≥3 (where ΔCq is the difference between the average Cq of the NTC and the lowest input), and a Quality Score ≥4 (based on linearity, reproducibility, curve shape, and other parameters). Primers targeting GAPDH variants (rat, mouse, tomato, yeast), PGK variants (rat), and B-actin variants (rat, mouse, tomato) were used with total RNA purified from rat brain, rat liver, rat kidney, mouse kidney, tomato leaf, and yeast as templates. RT-qPCR assays were assembled as directed using Luna RT-qPCR reagents, and cycled on a Bio-Rad CFX384. RT-qPCR on Monarch-purified RNA generates high-quality qPCR curves demonstrating accurate quantitation across a wide variety of sample types Monarch-purified RNA from human whole blood, rat kidney, tomato leaf, and the yeast S.cerevisiae was diluted to produce a five-log range of input template concentrations. Primers targeting GAPDH variants (tomato, yeast), PGK (rat), or SMG1 (human blood) were used for RT-qPCR assays, assembled as directed using Luna RT-qPCR reagents (NEB #E3005) and cycled on a Bio-Rad CFX384. Q: Are the Monarch Spin RNA Cleanup Kits compatible with Luna RT-qPCR reagents? A: Yes. RNA purified with these kits can be used for RT-qPCR with all of NEB’s Luna One-Step RT-qPCR Kits and the LunaScript® RT products. Q: Can I use shorter cycling times? A: Yes. For many targets, shorter incubation times can be used at each step. However, standard cycling conditions are recommended as a starting point, and may be required for challenging assays such as those with low DNA input.