New England Biolabs, E3025L, LunaScript® RT Master Mix Kit (Primer-free)

The LunaScript RT Master Mix Kit (Primer-free) features an optimized 5X master mix containing all the necessary components for first strand cDNA synthesis, except primers. Related Categories Luna® qPCR & RT-qPCR,, RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications RT-PCR,, cDNA Synthesis,, Reverse Transcription (cDNA Synthesis), FAQ Q: Can I use  RNA samples purified from different storage conditions such as RNALater in my LunaScript®  cDNA synthesis reactions? A: In general, purified RNA samples from different sources work well with LunaScript products. We typically recommend RNA purification using column-based methods (e.g., Monarch® RNA Miniprep Kit (NEB #T2010S)). Q: Can I set up my cDNA synthesis reaction at room temperature when using the LunaScript®  RT SuperMix? A: Yes. LunaScript cDNA synthesis reactions can be set up at room temperature. Q: Can I use LunaScript® cDNA products directly in qPCR? How much can I use for detection in a qPCR reaction? A: The cDNA product can be directly used in qPCR reaction. In general, we recommend 1 μl cDNA product in a 20 μl qPCR detection. When useful, up to 20% qPCR volume can be undiluted cDNA product (e.g. 4 μl cDNA product in a 20 μl qPCR reaction). Q: How much RNA template should I use in my LunaScript®  cDNA synthesis reactions? A: The LunaScript mixes 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 ≤ 1 μg In vitro transcripts 100 ng Q: How should I store LunaScript®  cDNA products? A: The cDNA product can be stored at 4°C for near term use. For long term storage, it should be stored at -20°C or -80°C. Q: How should I choose between a one-step RT-qPCR or two-step RT-qPCR approach? A: Two methods are available for quantifying RNA samples: one-step 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, where sample inputs may be limiting or numerous samples are examined. 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: Should I include a No-Reverse Transcriptase (RT) control reaction when setting up my cDNA synthesis reaction? A: Yes. When present, genomic DNA or carryover products can interfere with accurate RNA quantitation, particularly for low copy targets. Therefore, it is important to carry out the appropriate No-RT control reactions to account for these effects. In addition, no template control (NTC) reactions should be set up to demonstrate that positive reactions are meaningful. Please refer to LunaScript RT SuperMix Kit (NEB #E3010) should you need a No-RT Control mix. Q: What qPCR reagents do you recommend for detection of cDNA products? A: The cDNA products generated using the LunaScript® mixes have been extensively evaluated in qPCR using the Luna® Universal qPCR Master Mix (NEB #M3003) and the Luna Universal Probe qPCR Master Mix (NEB #M3004). In combination, these products provide a two-step RT-qPCR workflow with excellent sensitivity and accurate, linear quantitation. In addition, LunaScript is compatible with other major commercial qPCR mixes. Q: Why do I have low cDNA yields? A: There are several causes for low cDNA yield. Here are some suggestions to improve the yield: Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer (Agilent). Intact RNA of high purity is essential for full-length cDNA synthesis. RNA should have a minimum A260/A280 ratio of 1.7 or a RIN number greater than 8. Ethanol precipitation followed by a 70% ethanol wash can remove contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides. An RNA purification step using RNA extraction kits or phenol/chloroform extraction can remove contaminant proteins such as proteases. Increase the amount of RNA used, especially for low abundant RNA. Q: Will the blue dye in the LunaScript®  RT SuperMix interfere with detection? A: The LunaScript products contain an inert visual reference dye that gives the mix a clear blue color. This colored appearance makes it easier to track reaction setup by identifying the presence vs. absence and volume differences of cDNA reactions inside tubes, pipet tips, and downstream qPCR reactions. The blue dye has been extensively evaluated in cDNA synthesis and qPCR experiments and it has not been observed to inhibit or interfere with RNA detection. Q: How do I choose between the first strand cDNA synthesis kits from NEB? A: Table 1 highlights the major advantages and applications of the first strand cDNA synthesis kits from NEB. cDNA Synthesis Kits LunaScript® RT SuperMix Kit (NEB #E3010) LunaScript RT Master Mix Kit (Primer-free) (NEB #E3025) ProtoScript® II First Strand cDNA Synthesis Kit (NEB #E6560) Components LunaScript RT SuperMix (5X)* No-RT Control Mix (5X)* Nuclease-free Water *Both mixes contain hexamer and d(T)10 LunaScript RT Master Mix (Primer-free) (5X) No-RT Control Mix (Primer-free) (5X) Nuclease-free Water ProtoScript II Enzyme Mix (10X) ProtoScript II Reaction Mix (2X) Oligo d(T)23VN (50 µM) Random Primer Mix (60 µM) Nuclease-free Water Format Recommended application Primers Incubation For 2-step RT-qPCR detection Hexamer and d(T)10 included 25°C 2 min; 55°C 10 min; 95°C 1 min For 2-step RT-qPCR detection Random Primer Mix 25°C 2 min; 55°C 10 min; 95°C 1 min For general cDNA synthesis d(T)23VN or gene-specific primers 55°C 10 min; 95°C 1 min Random Primer Mix 25°C 2 min; 55°C 10 min; 95°C 1 min For general cDNA synthesis d(T)23VN or gene-specific primers 42°C 60 min; 80°C 5 min Random Primer Mix 25°C 5 min; 42°C 60 min; 80°C 5 min Advantages ✓ Supermix ✓ Short protocol ✓ A visible tracking indicator ✓ Higher reaction temperature ✓ Higher volume of RNA input ✓ Flexible choice of primers ✓ Short protocol ✓ A visible tracking indicator ✓ Higher reaction temperature ✓ Higher volume of RNA input ✓ Flexible choice of primers ✓ Everything included Follow the decision tree below to choose the first strand cDNA synthesis kit based on your downstream applications. Figure 1. LunaScript RT Master Mix (Primer-free) supports numerous applications. By adding different primers including Random Primer Mix, d(T)23VN, and random hexamers, cDNA produced by LunaScript RT Master Mix can be ideally suited for downstream applications such as RT-qPCR detection, RT-PCR, and RNA-seq studies. Q: When using LunaScript cDNA products in downstream PCR (i.e., two-step RT-PCR), what volumes and PCR products can be used? A: The cDNA products of LunaScript reactions are compatible with many PCR reagents. Typically, we recommend OneTaq 2X Master Mix (NEB #M0482 or NEB #M0485) for PCR detection up to 5kb, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) for highest fidelity, and LongAmp Taq 2X Master Mix (NEB #M0287) for high yields from longer products. Depending on the targets/primers, the optimal cDNA input may vary and should be optimized but typically, we recommend 1 μl of the cDNA reaction as input into a 25 μl PCR. Where useful, up to 20% PCR volume can be added (e.g. 5 μl cDNA product in a 25 μl PCR). Q: How do I choose primers for cDNA synthesis using LunaScript RT Master Mix (Primer-free)? A: The optimal primer for cDNA synthesis depends on the desired downstream application. Downstream Applications Primers Goal qPCR quantitation Random Primer Mix (NEB #S1330S) Typical recommendation: 6 µM final Even coverage General cDNA synthesis Oligo d(T)23VN (NEB #S1327S) Typical recommendation: 5 µM final Full-length cDNA synthesis Random Primer Mix or Random Primer Typical recommendation: 6 µM final Even coverage Gene-specific Primer Typical recommendation: 0.5 µM final (Range: 0.1 - 1 µM) Target-specific cDNA Q: Can I use LunaScript RT Master Mix in RNA-seq workflows? A: LunaScript RT Master Mix is compatible with different primers including random primers and oligo-dT primers. For most RNA-seq workflows, LunaScript RT Master Mix can be incorporated as the cDNA synthesis step. Q: What temperature should I use for my LunaScript®  cDNA synthesis reaction? A: The thermostable Luna® Reverse Transcriptase supports efficient cDNA synthesis at elevated temperatures compared to MMLV RT. For most targets, cDNA synthesis can be accomplished with a 10-minute incubation at 55°C. For difficult templates, the cDNA synthesis temperature can be increased up to 65°C. Q: How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products? A: Induro Reverse Transcriptase is a group II intron-encoded RT that is useful for cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. In contrast, the LunaScript reagents are master mixes. The LunaScript RT Master Mix Kit (Primer-free) (NEB #E3025) is suitable for general cDNA synthesis. LunaScript RT SuperMix products (NEB #E3010/M3010) are optimized for two step RT-qPCR workflows where only short cDNA products are required. LunaScript RT SuperMix is also featured in the ARTIC SARS-CoV-2 sequencing workflow. This table outlines the applications and features of each. Intron-encoded RT Retroviral RT Products Induro™ Reverse Transcriptase (NEB #M0681) LunaScript® RT SuperMix Kit (NEB #E3010) LunaScript RT SuperMix (NEB #M3010) LunaScript RT Master Mix Kit (NEB #E3025) ProtoScript® II First Strand cDNA Synthesis Kit (NEB #E6560) Applications Long cDNA synthesis Impure RNA samples RNA-seq applications (e.g., direct RNA sequencing, long-read cDNA sequencing, tRNA seq Applications 2-step RT-qPCR detection] RNA-seq workflow Applications RNA-seq workflow 2-step RT-qPCR detection General cDNA synthesis 2-step RT-PCR detection Applications General cDNA synthesis RNA-seq workflow 2-step RT-PCR detection ✓ Short protocol ✓ Flexible choice of primers, modified dNTPs ✓ Higher reaction temperature ✓ Supermix ✓ Short protocol ✓ Tracking dye ✓ Higher reaction temperature ✓ Flexible choice of primers ✓ Short protocol ✓ Tracking dye ✓ Higher reaction temperature ✓ Flexible choice of primers ✓ Kit format includes all necessary components Q: How do I know whether my template RNA is of good quality? A: Intact RNA of high purity is essential for full-length cDNA synthesis. An absorbance ratio at A260/A280 above 1.7 or RIN number greater than 8 on a BioAnalyzer usually indicates RNA of high quality.