DETAILED PRODUCT INFORMATION & DATA
55C MMLV-RT (MDX117)
Robust thermostable reverse transcriptase ideal for low copy number RNA targets with high secondary structure.
• Reverse transcriptase activity up to 60°C • Ideal for RNA with high secondary structure such as viral genomes • Sensitive detection of low copy number RNA targets • Formulation compatible with lyophilization and air-drying applications • Ideal for developing all of your assay needs
High Thermostability 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 a new 55C MMLV-RT (200 U/ µ L) 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. Performance of Meridian’s 55C MMLV-RT ( red ), standard MMLV-RT ( grey ) and a SuperScript III Reverse Transcriptase (Thermo, black ) after pre-incubation at 40°C to 70°C for 10 mins, in a multiplex one-step RT-qPCR assay. Δ 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.
Comparison of thermostability of 55C MMLV-RT vs other RTases
Higher Enzyme Efficiency and Sensitivity
55C MMLV-RT is designed for greater efficiency of the reverse transcription reaction, enabling a lower limit of detection (LOD) with higher sensitivity in one-step RT-qPCR assays.
The sensitivity of 55C MMLV-RT ( red ) was compared to SuperScript III Reverse Transcriptase ( 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.
Compatible with Lyophilization and Air-Drying Applications 55C MMLV-RT proprietary formulation allows for incorporation into assays designed for subsequent lyophilization or air-drying.
Dried Mix | Wet Mix
55C MMLV-RT was added to an air-dryable RT-qPCR Mix and dried down in a fan assisted oven ( red ). After rehydration, the mix was tested against freshly prepared (wet) version of this mix ( blue ), in a triplex one-step RT-qPCR assay on respiratory RNA virus targets (Influenza A, MERS-CoV and RSV). The results demonstrate that 55C MMLV-RT retains the same activity level after air-drying even in challenging conditions such as multiplex RT-qPCR reactions.
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