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产品信息
LunaScript RT SuperMix is an optimized master mix for first strand cDNA synthesis and can be used in amplicon sequencing or a two-step RT-qPCR workflow. It features the thermostable Luna Reverse Transcriptase, which supports cDNA synthesis at elevated temperatures. Murine RNase Inhibitor is also included to protect template RNA from degradation. LunaScript RT SuperMix contains random hexamer and poly-dT primers, allowing even coverage across the length of the RNA targets. In addition, LunaScript RT SuperMix contains a blue dye, providing a visual indicator that can be followed throughout the two-step RT-qPCR process. The product offers robust, linear, and sensitive detection using total RNA inputs as high as 1 µg and as low as single copies of RNA.
For RT-PCR, RNA-seq, and other applications, LunaScript RT Master Mix Kit (Primer-free) (NEB #E3025) is an optimized master mix containing all the necessary components for first strand cDNA synthesis, except for primers. The mix is compatible with random primers, oligo dT primers, and gene-specific primers, enabling maximum cDNA synthesis flexibility. Learn more here.
In comparison to the LunaScript kit version (NEB #E3010), this LunaScript product does not contain a No-RT Control Mix or nuclease-free water. More details can be found below:
Figure 1: Comparison of LunaScript Products
Figure 2: Product options for one-step and two-step RT-qPCR workflows
Figure 3: LunaScript RT SuperMix offers exceptional sensitivity, linearity and reproducibility in two-step RT-qPCR workflows
RNA was converted to cDNA using 1X LunaScript RT SuperMix in 20 μl reactions using standard reaction conditions (25°C/2 min, 55°C/10 min, 95°C/1 min). cDNA was then quantitated by qPCR using Luna Universal qPCR Master Mix (NEB #M3003) and 1 μl of cDNA product as template, with triplicate reactions at each input concentration.
A. A serial dilution of Jurkat total RNA (1 μg – 1 pg) was converted to cDNA and then quantitated by qPCR using a β-actin target.
B. ERCC (External RNA Controls Consortium) mix1 RNA containing 5×109 to 50 copies of ERCC00130 (~10 ng – 10 fg) was converted to cDNA and then quantitated by qPCR.
Figure 4: LunaScript RT SuperMix supports even coverage of long transcripts
Eight pairs of primers were designed to cover the entire 15.2 kb HERC1 transcript. Jurkat total RNA (1 μg – 1 ng) was converted to cDNA using 1X LunaScript RT SuperMix in 20 μl reactions using standard reaction conditions (25°C/2 min, 55°C/10 min, 95°C/1 min). cDNA coverage was then evaluated by qPCR using Luna Universal qPCR Master Mix (NEB #M3003) and 1 μl of cDNA product as template, with duplicate reactions for each target at each input concentration.Figure 5: LunaScript RT SuperMix demonstrates superior linear detection of RNA targets
Commercially available cDNA supermixes were used according to manufacturer’s recommendations to generate cDNA from 1 μg – 100 pg human (Jurkat) total RNA. cDNA products were then evaluated by qPCR using eight targets varying in abundance, length and %GC. qPCR detection was performed using Luna Universal qPCR Master Mix (NEB #M3003) or Luna Universal Probe qPCR Master Mix (NEB #M3004). Results were evaluated for efficiency and ΔCq, where ΔCq measures low input detection and lack of non-template control (NTC) amplification (ΔCq = average Cq of NTC – average Cq of lowest input). Green box indicates target performance criteria (Efficiency = 90-110%, ΔCq ≥ 3).
Learn more about this “Dots in Boxes” visualization method.
Figure 6: At just 13 minutes, LunaScript RT SuperMix offers the shortest available first-strand cDNA synthesis protocol
Comparison of recommended protocols for cDNA synthesis. LunaScript RT SuperMix requires the shortest reaction time and tolerates elevated temperatures, reducing complications from RNA secondary structure.Figure 7: LunaScript RT SuperMix exhibits exceptional stability at room temperature
LunaScript RT SuperMix and other commercial products were left at 25°C for 15 days, and then used according to manufacturer’s recommendations to generate cDNA from 1 μg – 100 pg human (Jurkat) total RNA. cDNA products were then evaluated by qPCR using eight targets varying in abundance, length and %GC. qPCR detection was performed using Luna Universal qPCR Master Mix (NEB #M3003). Results were evaluated for efficiency and ΔCq, where ΔCq measures low input detection and lack of no-template control (NTC) amplification (ΔCq = average Cq of NTC – average Cq of lowest input). Green box indicates target performance criteria (Efficiency = 90-110%, ΔCq ≥ 3).
Learn more about this “Dots in Boxes” visualization method.
产品类别:
Luna® qPCR & RT-qPCR Products
应用:
qPCR & RT-qPCR,
Reverse Transcription (cDNA Synthesis)
试剂盒组成
试剂盒组成
本产品提供以下试剂或组分：
NEB #
名称
组分货号
储存温度
数量
浓度
E3010S
-20
LunaScript® RT SuperMix
M3010SVIAL
-20
1 x 0.1 ml
5 X
No-RT Control Mix
M3011SVIAL
-20
1 x 0.1 ml
5 X
Nuclease-free Water
B1502AVIAL
-20
1 x 1.5 ml
不适用
E3010L
-20
LunaScript® RT SuperMix
M3010LVIAL
-20
1 x 0.4 ml
5 X
No-RT Control Mix
M3011LVIAL
-20
1 x 0.4 ml
5 X
Nuclease-free Water
B1502AVIAL
-20
4 x 1.5 ml
不适用
特性和用法
相关产品
相关产品
Luna® Universal qPCR Master Mix
Luna® Universal Probe qPCR Master Mix
Antarctic Thermolabile UDG
Monarch® Total RNA Miniprep Kit
操作说明、说明书 & 用法
操作说明
Protocol for LunaScript® RT SuperMix Kit (E3010)
Protocol for Two-step RT-qPCR using the LunaScript® RT SuperMix Kit (NEB #E3010) and the Luna® Universal qPCR Master Mix (NEB #M3003) or Luna Universal Probe qPCR Master Mix (NEB #M3004)
说明书
产品说明书包含产品使用的详细信息、产品配方和质控分析。
manualE3010
应用实例
Facilitating Detection of SARS-CoV-2 Directly from Patient Samples: Precursor Studies with RT-qPCR and Colorimetric RT-LAMP Reagents
FAQs & 问题解决指南
FAQs
Can I use RNA samples purified from different storage conditions such as RNALater in my LunaScript® cDNA synthesis reactions?
Can I add gene-specific primers in my LunaScript® cDNA synthesis reaction?
Can I set up my cDNA synthesis reaction at room temperature when using the LunaScript® RT SuperMix?
Can I use a shorter incubation time for my cDNA synthesis reaction when using the LunaScript® RT SuperMix?
Can I use LunaScript® cDNA products directly in qPCR? How much can I use for detection in a qPCR reaction?
How much RNA template should I use in my LunaScript® cDNA synthesis reactions?
How should I store LunaScript® cDNA products?
How should I choose between a one-step RT-qPCR or two-step RT-qPCR approach?
Should I include a No-Reverse Transcriptase (RT) control reaction when setting up my cDNA synthesis reaction?
What qPCR reagents do you recommend for detection of cDNA products?
What temperature should I use for my LunaScript® cDNA synthesis reaction?
Why do I have low cDNA yields?
Will the blue dye in the LunaScript® RT SuperMix interfere with detection?
How do I choose between the first strand cDNA synthesis kits from NEB?
When using LunaScript cDNA products in downstream PCR (i.e., two-step RT-PCR), what volumes and PCR products can be used?
Can I use LunaScript RT SuperMix in RNA-seq workflows?
How do I choose between LunaScript® RT SuperMix Kit and ProtoScript first strand cDNA synthesis kits?
Can I use a shorter incubation time for my cDNA synthesis reaction when using the LunaScript® RT Master Mix (Primer-free)? Does longer incubation increase the yield of cDNA?
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