Reverse Transcription Kit

• Yields high full-length cDNA products (up to 10 kb)
• Possesses increased sensitivity in RT-qPCR and RT-PCR assays
• Requires fewer pipetting steps for minimized contamination risk
• Shows no RNase H or 3’→5’ exonuclease activity
• Has supreme thermostability and is suitable for complex RNA templates

The Reverse Transcription Kit contains all the necessary components for synthesizing first-strand cDNA from the total RNA or mRNA templates. The synthesized single-stranded cDNA is suitable for real-time quantitative RT-PCR applications. The kit provides high yields of full-length cDNA products and increases RT-qPCR sensitivity. Starting material can range from 10 pg to 5 μg of total RNA.

The kit includes a recombinant thermostable reverse transcriptase M-MuLV without RNase H activity and the RNase Inhibitor HU. The optimal reaction buffer with a combination of random hexamers, oligo (dT)18 primers, and MgCl₂ and dNTPs mix increases sensitivity.

Reverse Transcription Kit has increased thermal stability, which allows the reaction to be carried out at a higher temperature (optimum activity at 50°C). 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 up to 10 kb long.

Using the recombinant RNase inhibitor HU protects the RNA template from degradation. RNase H selectively hydrolyzes the phosphodiester bonds of RNA only when hybridized into DNA. The RNase H does not degrade either single and double-stranded DNA or unhybridized RNA. This optional step can improve the sensitivity of subsequent RT-qPCR reactions since the PCR primers will bind more easily to the cDNA.

The RNase H is recommended only when it contributes to full-length cDNA synthesis and increased yields of first-strand cDNA.

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.
• DNase I 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 reaction mixture preparation, keep all kit reagents on ice or in a freezing rack.

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

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

The activity of Reverse Transcriptase Kit is inhibited by metal ion chelating agents (e.g., EDTA), inorganic phosphor, pyrophosphate and polyamines.

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.