Robust thermostable reverse transcriptase ideal for ambient temperature stable molecular assays.
R everse transcriptase activity up to 60°C
Ideal for RNA with high secondary structure such as viral genomes
S ensitive detection of low copy number RNA targets
Ideal for developing fast, highly reproducible RT-qPCR assays
Challenges with RNA Secondary Structures
55C MMLV-RT Thermostable Moloney Murine Leukemia Virus (concentration 200 U/µL)
Traditional Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) is not thermostable and can only maintain its enzymatic activity at relatively low temperatures (up to 50°C). However for cDNA synthesis, a higher reaction temperature is desirable as it reduces RNA secondary structures which can inhibit reverse transcription and it minimizes nonspecific primer binding. Meridian has developed 55C MMLV-RT a new reverse transcriptase that has higher thermal stability and reduced RNase H activity. The enzyme can be used to synthesize first-strand cDNA at temperatures up to 60°C, which improves the cDNA yield from difficult RNA targets that require higher temperature to denature strong secondary structures.
Comparison of thermostability of 55C MMLV-RT vs other RTases
Performance of Meridian’s 55C MMLV-RT (red) , standard MMLV-RT (grey) and SuperScript III thermostable reverse transcriptase (Thermo, black) after pre-incubation at 40°C to 70°C for 10 mins, in a multiplex one-step RT-qPCR assay. ∆ ct values were calculated against the ct value produced by the same enzyme stored at -20 °C. The data illustrates the increased thermal stability of 55C MMLV-RT when compared to other MMLV-RT enzymes and its ability to efficiently synthesize cDNA at temperatures up to 60°C.
MMLV-RT | SuperScript III (Thermo) | 55C MMLV-RT
Higher Enzyme Efficiency and Sensitivity
55C MMLV-RT is designed for greater efficiency of the reverse transcription reaction, improving cDNA yield and enabling a lower limit of detection (LOD) with higher sensitivity in one-step RT-qPCR assays.
55C MMLV-RT | SuperScript III (Thermo)
The sensitivity of 55C MMLV-RT (red) was compared to SuperScript III (Thermo, black) in a multiplex one-step RT-qPCR assay using a 10-fold serial dilution of mammalian total RNA. The results demonstrate that 55C MMLV-RT has higher performance with better sensitivity and end-fluorescence.
Ideal for High-Throughput, Multiplex One-Step RT-qPCR Assays
55C MMLV-RT exhibits robust performance in one-step RT-qPCR assays detecting RNA viruses with strong secondary structures.
55C MMLV-RT was tested in a triplex one-step RT-qPCR assay on respiratory RNA virus targets (Influenza A, MERS-CoV and RSV). The results demonstrate the sensitivity and reproducibility of 55C MMLV-RT even in challenging conditions such as multiplex RT-qPCR reactions where targets may differ greatly in abundance.
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