Meridian is a primary manufacturer of specialized high-quality molecular reagents and offers solutions to a wide range of industries to diagnose and treat diseases, discover new therapeutics or develop tests for environmental, food and cosmetic safety.
Reverse transcriptases High-performance RTases for cDNA Synthesis
Reverse transcriptases are essential for synthesizing complementary DNA (cDNA) strands from RNA templates. They direct the synthesis of first strand cDNA, which can be used directly as a template for applications such as PCR, qPCR, LAMP, or sequencing workflows. There are several key important features when determining the best RTase for your application: 1. RNase H activity: RNase H (ribonuclease H) is an enzyme that catalyzes the cleavage of the RNA strand in an RNA-DNA complex and degrades the RNA, while leaving the DNA strand intact. However, if the RNAse H activity of RTases is too high, it can lead to excessive degradation of the RNA template, resulting in incomplete or inefficient cDNA synthesis. RTases with low RNAse H activity minimize the nonspecific degradation of RNA molecules in the reaction, improving the specificity and fidelity of cDNA synthesis. 2. Processivity: Processivity is defined by the number of nucleotides incorporated in a single binding event. It is also important for creating long transcripts in a short reaction time. Most engineered reverse transcriptases have improved processivity, (up to 65x greater than wild-type MMLV reverse transcriptases). 3. Thermostability: The ability to withstand high temperatures is an important aspect of cDNA synthesis, as high temperatures help denature RNA that has strong secondary structures and/or high GC content enabling full-length cDNA synthesis and high yields. Wild-type MMLV-RT performs optimally at 37°C whereas engineered thermostable MMLV-RTs can withstand temperatures up to 55°C without negatively impacting reverse transcription efficiency. Meridian offers several RTs, each containing unique product features such as high thermostability or lyophilization compatibility for creating room-temperature stable mixes. View our selection chart for details:
One Step RT-qPCR/ RT-LAMP
Two Step RT-qPCR/ RT-LAMP
Thermostability
Name / Features
Conc.
Cat. No
55C MMLV-RT Compatible with lyophiliztion or air-drying
MDX117
Up to 60°C
200 U/ µ L
RNase-Tolerant MMLV-RT Contains an RNAse inhibitor to improve reaction efficiency
MDX043
Up to 50°C
100x
MMLV-RT Lower RNase H activity
MDX044
100x
Up to 45°C
Lyo-Compatible MMLV-RT Glycerol-free formulation
MDX042
100x
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DETAILED PRODUCT INFORMATION & DATA
55C MMLV-RT (MDX117)
Robust thermostable reverse transcriptase ideal for low copy number RNA targets with high secondary structure.
Features
• 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
Performance Data
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
B2MG
Rps18L
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.
Flu A
MERS-CoV
RSV
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.
Lyo-Compatible MMLV-RT (MDX042)
High concentration, glycerol-free reverse transcriptase ideal for ambient temperature stable molecular assays.
Features
• Glycerol free, ideal for lyophilization and air-drying applications • Reverse transcriptase activity up to 45°C • High-quality, first-strand cDNA synthesis most one-step RT-PCR and RT-qPCR applications • Sensitive detection of low copy number RNA targets
Performance Data
Higher Enzyme Efficiency and Sensitivity MMLV-RT is an unstable enzyme, so glycerol is normally added as it is an osmolyte, stabilizing the protein and preventing crystal formation during freezing, however, high glycerol concentrations are not compatible with drying down assays. The high concentration of Lyo-Compatible MMLV-RT overcomes these stability issues, allowing the glycerol to be removed and in turn allowing assays to be dried. Lyo-Compatible MMLV-RT is designed for multiplex RT-qPCR assays, allowing high sensitivity detection of low copy number RNA targets.
Hepatitis virus (RNA)
Cytomegalovirus (DNA)
The high efficiency of Lyo-compatible MMLV-RT when used with Lyo-Ready qPCR Mix (Cat# MDX021) in multiplex RT-qPCR assays with Hepatitis virus (RNA) and Cytomegalovirus (DNA) allows for high sensitivity detection of low copy number targets (as few as five viral particles).
RNase-Tolerant MMLV-RT (MDX043)
Reverse transcriptase/ RNase inhibitor mix optimized for one-step RT-qPCR.
Features
• High efficiency where samples are likely to contain inhibitors • Reverse transcriptase activity up to 50°C • Sensitive detection of low copy number RNA targets • High-quality, first-strand cDNA synthesis ideal for one-step RT-qPCR
Performance Data
High sensitivity RNase-Tolerant MMLV-RT is a 100x reverse transcriptase/RNase inhibitor mix optimized for one-step RT-PCR. RNases are ubiquitous and it is very easy to contaminate samples during reaction setup or sample preparation, even traces of RNase can be detrimental, nicking the RNA and causing shortened cDNA products, low yields, and reduced RT-PCR sensitivity. The RNase Inhibitor in RNase-Tolerant MMLV-RT is a highly efficient inhibitor of a broad spectrum of eukaryotic RNases and shows no inhibition of the reverse transcriptase or polymerase activity. It can be used with a broader temperature range, for the detection of RNA at very low levels, making it ideal for applications such as blood bank or transplant viral testing. Sensitive detection of viruses from purified blood extracts HIV-2 (5 IU/mL) HAV (5 IU/mL)
24 replicates of purified blood viral extracts were tested for HIV-2 and HAV using Low LOD 1-Step RT-qPCR Mix (Cat# MDX025) and RNase-Tolerant MMLV-RT in multiplex RT-qPCR assays. The results demonstrate the sensitivity of the RNase-Tolerant MMLV-RT, allowing high sensitivity detection of low copy number targets (as few as five viral IU/mL (international units per millilitre)).
www.meridianbioscience.com/lifescience
MMLV-RT (MDX044)
Reverse transcriptase, ideal for most RT-PCR and RT-qPCR applications.
Features
• Lower RNase H activity for high yield • Reverse transcriptase activity up to 45°C • Sensitive detection of low concentrations of template RNA • High-quality, first-strand cDNA synthesis ideal for most RT-PCR and RT-qPCR applications
Performance Data
High sensitivity MMLV-RT is a 100x high-quality reverse transcriptase that synthesizes a complementary DNA (cDNA) strand from mRNA or total RNA. With higher thermostability and reduced RNase H activity, it can be used for synthesis with long messenger RNA templates with high sensitivity and efficiency and is ideal for detecting low-level target genes. Sensitive transcription, of a 1 kb fragment of calnexin from down to 10pg total RNA
A 5-fold serial dilution of total RNA (1 μg to 10 pg) was reverse transcribed using MMLV-RT, and the resultant cDNA was then used as a template in a PCR with primers for amplification of a 1 kb fragment from calnexin. The results demonstrate the high sensitivity of MMLV-RT even when the amount of template RNA is limited.
Ordering Information
PRODUCT
CAT NO.
VOLUME
REACTIONS 10,000 Units 40,000 Units 200,000 Units 2,000,000 Units 10,000,000 Units 1,000 Reactions 10,000 Reactions 1,000 Reactions
50 µ L
200 µ L
55C MMLV-RT
1 mL
MDX117
10 mL 50 mL
8 µ L
Lyo-Compatible MMLV-RT
MDX042
80 µ L
200 µ L
RNase Tolerant MMLV-RT
2 mL 10,000 Reactions 210 mL 50,000 Reactions
MDX043
200 µ L
1,000 Reactions 10,000 Reactions
MMLV-RT
MDX044
2 mL
Ordering information: USA 5171 Wilfong Road Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223
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