StableScript Reverse Transcriptase

• OEM by QIAGEN offers bulk manufacturing of StableScript Reverse Transcriptase in custom formulations
• One-step qRT-PCR and long-range RT-PCR
• Highly sensitive RNA detection
• Ask about low-glycerol formulations

StableScript is a versatile reverse transcriptase designed for use in one-step qRT-PCR and long-range RT-PCR. The enzyme demonstrates high sensitivity for RNA detection, improved thermostability, processivity and inhibitor resistance over the first generation M-MLV reverse transcriptase RNase H minus. >

The enzyme is supplied in 20 mM K₃P0₄ (pH 7.0), 1 mM EDTA, 1 mM DTT, stabilizer and 50% glycerol.

Ask about low glycerol formulations.

StableScript Reverse Transcriptase is a versatile reverse transcriptase designed for use in one-step RT-qPCR and long-range RT-PCR. The enzyme demonstrates high sensitivity for RNA detection, improved thermostability, processivity and inhibitor resistance over first generation M-MLV reverse transcriptase RNase H minus.

Properties:

• Optimum extension temperature: 55°C
• Transcription length: 12.3 kb
• Storage temperature: –15°C to –25°C
• Extension temperature range: 50°C to 65°C

Details about StableScript Reverse Transcriptase performance can be see in figures “ Temperature profile – active from 45°C to 65°C – optimal 55°C”, “ Increased thermostability helps to overcome difficult RNA targets because RNA structures unravel and are less likely to prevent amplification.”, “Alkaline gel-electrophoresis.”, “Long-range PCR.”, “StableScript maintains performance across a wide pH range.”, “ Robust RT-qPCR performance in the presence of common inhibitors.”, “Superior sensitivity and dynamic range in one-step RT-qPCR.”, “Similar performance in lyophilized samples.” and “Exceptional lot-to-lot consistency.”

Details about StableScript Reverse Transcriptase performance can be see in figures “Temperature profile – active from 45°C to 65°C – optimal 55°C”, “ Increased thermostability helps to overcome difficult RNA targets because RNA structures unravel and are less likely to prevent amplification.”, “Alkaline gel-electrophoresis.”, “Long-range PCR.”, “ StableScript maintains performance across a wide pH range.”, “ Robust RT-qPCR performance in the presence of common inhibitors.”, “Superior sensitivity and dynamic range in one-step RT-qPCR.”, “Similar performance in lyophilized samples.” and “Exceptional lot-to-lot consistency.”

Source of recombinant enzyme protein

The protein is produced by a recombinant E. coli strain carrying engineered reverse transcriptase gene from equine infectious anemia virus. This novel reverse transcriptase has no detectable RNase H activity and has increased thermostability.

Unit definition

One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 30 minutes at 55°C using poly r(A)/oligo (dT) as a substrate.

Quality control analysis
Unit activity is measured using a twofold serial dilution method. Dilutions of enzyme were made in 1x StableScript Diluent and added to 50 µL reactions containing 20 µg/mL poly r(A) RNA, oligo (dT) DNA, 1x StableScript Reaction Buffer, 3H-dTTP and 250 µM dTTP and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

The functionality of the RT-PCR assay is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The amplification threshold (C_q) of the test lot is compared to a reference lot.

Protein concentration (OD₂₈₀) is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein-of-interest band in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for four hours at 37°C.

Double-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL enzyme solution incubated for four hours at 37°C.

Double-stranded endonuclease is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL enzyme solution that is incubated for four hours at 37°C.

E. coli 16S rDNA contamination is evaluated using 5 µL replicate samples of enzyme solution denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert 1-Kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• RT-qPCR
• cDNA synthesis
• Long-range RT-PCR