Reverse Transcriptase

• Yields high full-length cDNA synthesis (up to 7 kb)
• Possesses increased sensitivity in RT-qPCR and RT-PCR assays
• Shows no RNase H or 3’→5’ exonuclease activity
• Has supreme thermostability and is suitable for complex RNA templates

Reverse Transcriptase is a modified, recombinant form of the Reverse Transcriptase from the Moloney Murine Leukemia Virus (M-MuLV) purified from Escherichia coli. Reverse Transcriptase synthesizes a complementary DNA strand in the presence of a primer using either RNA (cDNA synthesis) or single-stranded DNA (ssDNA) as a template.

Reverse Transcriptase has increased thermal stability, which allows the reaction to be carried out at a higher temperature (optimum activity at 50°C).

It is supplied with 10x RT Reaction Buffer containing 500 Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl₂, 100 mM DTT.

One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into an acid-insoluble material in 10 minutes at 37°C, using poly(A) x oligo(dT)12-18 as template primer.

It increases the efficiency and specificity of those transcribed RNA regions rich in GC pairs and containing secondary structures. The enzyme has no 3’ – 5’ exonuclease or RNase H activity, which improves the synthesis of a full-length cDNA, even from long mRNA templates, using random priming. The enzyme gives high yields of first-strand cDNA synthesis up to 7 kb long.

Working with RNA

Acquiring high-quality, intact RNA, free of genomic DNA and RNase traces, is vital for synthesizing a full-length cDNA followed by an accurate quantitative analysis (qPCR).

The following recommendations should be followed:

• Maintain aseptic working conditions by using disposable gloves and changing them as frequently as required; use RNase-free consumables; only work in an area assigned for working with RNA and with equipment designated for that purpose.
• dsDNase HL enzyme (not included) may be used if obtaining a DNA-free RNA sample is required.
• RNA samples should be stored aliquoted at -70°C. Avoid subjecting the samples to repeated freezing and thawing cycles.

Additional information

During RT-PCR preparation, keep Reverse Transcriptase and 10x RT Reaction Buffer on ice or in a freezing rack.

Use an RNase H treatment for reactions sensitive to residue RNA traces to increase the sensitivity of RT-qPCR.

The quantity of cDNA used when preparing PCR or qPCR reactions should not exceed 1/10 of a final reaction volume; e.g., a maximum volume of 2.5 µL of cDNA should be used in a 25 µL reaction.

Metal ion chelating agents inhibit the activity of Reverse Transcriptase (e.g., EDTA), inorganic phosphors, pyrophosphates and polyamines.

Enzyme inactivation should be carried out at 85°C for 5 minutes.

Quality Control

Protein purity is determined using assay by SDS-PAGE electrophoresis resulting in >90% purity.

RNase and DNase contamination is evaluated by assessing RNase and DNase activity. In addition, functional quality is tested by RT-PCR experiment.

This is used for applications such as:

• Full-length cDNA template synthesis for RT-qPCR and two-step RT-PCR assays
• cDNA synthesis for molecular cloning
• cDNA library construction
• RNA analysis

The acquisition of BLIRT by QIAGEN in the second quarter of 2022 paved the way for BLIRT’s expansion of R&D and manufacturing capabilities for customized and standardized recombinant enzymes and molecular biology reagents. Due to the transition, the rebranding process from BLIRT to QIAGEN is ongoing and the following resources still show BLIRT branding.
 
We assure you that the integration does not affect the quality, fit, form and function of the products.