The LunaScript RT Master Mix Kit (Primer-free) includes an optimized 5X master mix containing all the necessary components for first strand cDNA synthesis, except primers. The mix features the thermostable Luna Reverse Transcriptase, which supports cDNA synthesis at elevated temperatures. Murine RNase Inhibitor is included to protect template RNA from degradation, along with dNTPs. In addition, the presence of a blue tracking dye provides a visual indicator for the RT step and downstream applications.
The mix is compatible with random primers, oligo dT primers, and gene-specific primers, enabling maximum cDNA synthesis flexibility. The cDNA product generated by the LunaScript RT Master Mix (Primer-free) can be used in a variety of downstream applications. For real-time PCR quantitation, a mixture of random and dT primers is recommended to generate cDNA. The mix offers robust, linear and sensitive detection from 1 μg total RNA input down to low copies of RNA, similar to the LunaScript RT SuperMix Kit (NEB #E3010). To generate long or full-length cDNAs, oligo dT primers should be used. With a short incubation at 55°C for 10 minutes, the LunaScript RT Master Mix (Primer-free) can make cDNA products up to 9 kb. A No-RT control mix is provided for ease of setting up control reactions.
Figure 1: Comparison of supermix, master mix and cDNA synthesis kit formats
Figure 2: At just 13 minutes, LunaScript offers the shortest available first-strand cDNA synthesis protocol
Comparison of recommended protocols for cDNA synthesis. Both LunaScript RT SuperMix Kit and LunaScript RT Master Mix Kit require the shortest reaction time and tolerate elevated temperatures, reducing complications from RNA secondary structure.
By adding different primers including a Random Primer Mix, d(T))23VN oligos, or random hexamers, the LunaScript RT Master Mix can produce cDNA that is ideally suited for downstream applications such as RT-qPCR, RT-PCR, and RNA-seq studies.
A. In the presence of Random Primer Mix (NEB #S1330), the LunaScript RT Master Mix (Primer-free) can deliver linear cDNA yields across RNA inputs from 1 µg to 100 pg with linearity similar to LunaScript (NEB #E3010) in a 2-step RT-qPCR workflow.
B. For average transcripts, the use of d(T)23VN primers offers cDNA coverage similar to the Random Primer Mix. However, for long transcripts (e.g., SMG1), random primers often enable better coverage of the 5´ end of the target, as seen in the example in the lower righthand box where the RT-qPCR target was located at the 5´ end of the 16 kb SMG1 transcript; its detection by an anchored dT primer alone [d(T)23VN] gave delayed Cq values when compared to the performance using the Random Primer Mix (a mixture of random hexamers and anchored dT primer).
Figure 5: Better performance of LunaScript RT Master Mix (Primer-free) in 2-step RT-qPCR quantitation
A. Several first strand cDNA synthesis kits were used according to manufacturer’s recommendations to generate cDNA using the random primers provided with each product. Input included human total RNA from 1 µg – 100 pg. cDNA products were then evaluated by qPCR using eight targets varying in abundance, length and %GC. qPCR detection of the cDNA products was performed using the 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). More details on this visualization method can be found in the application note “Development of a high-throughput data analysis method for quantitative real-time PCR (qPCR).”
B. Results from four targets above (two typical control genes and two others) were plotted to examine Cq as a function of input. LunaScript gave the earliest Cq values when compared to other commercial kits.
Figure 6: LunaScript RT Master Mix (Primer-free) can generate a broad range of cDNA products for use in routine or high fidelity PCR
LunaScript RT Master Mix (Primer-free) was used to synthesize cDNA from 1 µg of Jurkat total RNA in the presence of d(T))23VN primers under the standard reaction condition of 55°C for 10 min, 95°C for 1 min. Subsequent to cDNA synthesis, different PCR amplification reagents can be used for PCR detection. For routine application up to 5 kb, OneTaq 2X Master Mix (NEB #M0482) is recommended. For highest fidelity, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) is recommended. For highest yield of long PCR amplification, LongAmp Taq 2X Master Mix (NEB #M0287) can be used (data not shown).
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