RapiDxFire Thermostable Reverse Transcriptase

RapiDxFire Thermostable Reverse Transcriptase is a significant advancement over popular MMLV- and AMV reverse transcriptases. Originally identified in hot springs, this robust bacteriophage enzyme exhibits increasingly high activity at elevated temperatures (~60% activity remains after 10 min at 90 ⁰C) and maintains its core activity after >3 months storage at 20-25⁰C. This enzyme lacks RNAse H and 3’ 5’ exonuclease activity and efficiently synthesises short cDNA fragments (≤ 1 Kb). Like other reverse transcriptases, RapiDxFire RT possesses DNA polymerase activity but lacks 5′ 3′ exonuclease activity. Supplied in a glycerol-free, Triton™ X-100-free storage buffer, the enzyme formulation can be further optimised for downstream lyophilisation.

RapiDxFire Thermostable RT Performs Best at High Temperatures

Figure 1. Reaction temperature profile for RapiDxFire Thermostable Reverse Transcriptase. Reverse transcription reactions were set up on ice using each manufacturer’s recommended buffer system and Poly (rC) /p(dG)12-18 template/primer substrate. Reactions were transferred from ice to the indicated temperatures (37, 50, 55, 60, 65, 70, 75, and 80 °C) and incubated for 40 minutes. Following incubation, RNA/cDNA heteroduplex product is quantified as a measure of polymerisation activity, utilising PicoGreen® fluorescence on a Tecan Infinite® M1000 Pro. RapiDxFire Thermostable RT exhibits increasing activity as the reaction temperature is increased, up to 80 °C (highest temperature tested); ~60% activity remains after 10 min at 90 °C (data not shown).

Superior Detection of Zika virus with RapiDxFire Thermostable RT

Figure 2. cDNA synthesis time course studies in a 2-step RT-qPCR reaction with different thermostable RTs. A) qPCR curve after a 1 minute reverse transcription reaction. Zika cDNA synthesis was conducted for each enzyme in duplicate using target-specific primers and recommended reaction buffer and incubation temperatures per suppliers’ guidelines (RapiDxFire= 60 ⁰C, Supplier B= 50 ⁰C, and Supplier T= 55 ⁰C). After cDNA synthesis real time PCR was performed using one-tenth volume of each of the cDNA samples. PCR was performed using EconoTaq and its supplied buffer (Item No. 30031-3). Detection of PCR products were done real time using an intercalating dye (Dyomics Cat No. V13-01184) and BioRad CFX C1000 Touch™ with absorption/emission of 481nm/526nm. B) Reverse transcription time course study prior to performing second-step real-time PCR. Encircled data points are derived from data represented in the qPCR curve in A).

3-month Room Temperature Stability

Figure 3. RapiDxFire Thermostable RT was stored in separate aliquots per time point at ambient and -20 °C. At each time point RapiDxFire Thermostable RT was evaluated by measuring first-strand cDNA synthesis of 10000 copies MS2 RNA. cDNA synthesis was carried out using a gene-specific primer for MS2 at reaction temperature of 60 °C for 5 minutes. cDNA was measured using real time PCR and MS2 primers design around a 520 base pair amplicon.

Legal Information/Updates:
US Patent 8093030 for RapiDxFire™ is owned by LGC, Biosearch Technologies. For research use only. Not for use in diagnostic procedures.

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Key features

• Extremely active at high temperatures (55 °C to 80 °C): Improves specificity of cDNA Synthesis from diverse RNA templates
• Sensitive: Detects 100 copies of RNA in two-step RT-qPCR assays
• Short reaction time (5 minutes or less): Streamlined RT-qPCR workflow and faster time to results
• Stable at room temperature (> 3 months): Simplifies setup on automation decks (no cold storage required) and enables use in environments with limited cold-storage
• Lyo-compatible: Enzyme formulation is free of glycerol -and other components- that are known to interfere with downstream lyophilisation
• Batch to batch reproducibility: Manufactured in an ISO 13485-certified facility

Specifications

Storage: Store at -20 °C.

Storage Buffer: RapiDxFire Thermostable RT is supplied in a glycerol-free, Triton X-100-free buffer.

Unit Definition: One RT unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into an acid-insoluble form in 10 minutes at 55 °C (using poly(rA) oligo(dT) as template primer and 1X kit -supplied buffer formulation).

Functional QC Assay: RapiDxFire Thermostable RT is functionally tested with gene-° primer and bacteriophage MS2 RNA. In this reaction the enzyme converts 1000 copies of MS2 RNA into 520 bp cDNA in 5 minutes at 60 °C.

Endonuclease: Free of contaminating endonucleases at the 10 Unit level.

Exonuclease: Free of contaminating exonucleases at the 10 Unit level.

RNase Activity: No RNase activity detected using a fluorescence-based RNase activity assay.

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Product Details

Contents include RapiDxFire Thermostable Reverse Transcriptase (3 U/µL) and 10X RapiDxFire Reaction Buffer. The enzyme pack sizes (50 or 250 Rxns) are based on 25 µL reaction volumes.

Resources

Brochures and guides

app note rapidxfire reverse transcriptase

Documents

• 30250 RapidxFire Thermostable RT
• RapiDxFire reverse transcriptase manual
• app note rapidxfire reverse transcriptase