OneScript® Hot Reverse Transcriptase
| Cat. No. | G593 | |||||||||
| Name | OneScript® Hot Reverse Transcriptase | |||||||||
| Unit | 100 rxn | |||||||||
| Category | Reverse Transcriptase & RT-PCR | |||||||||
| Description |
OneScript® Hot Reverse Transcriptase is a thermostable, mutational derivative of Moloney-Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) that can efficiently reverse transcribe low-abundance or partially degraded RNA, and has significantly better resistance to contaminating inhibitors such as reagents used during RNA extraction and contaminants from biological samples. It possesses genetic modifications to abolish RNase H activity to achieve thermal stability, allowing for high processivity and sensitivity that enable rapid synthesis of full-length cDNA fragments in a fraction of the time required by leading competitors. abm is the only company in the world to have a thermostable reverse transcriptase engineered to offer superior cDNA synthesis performance with even the most challenging RNA samples due to its incredible thermostability at 60-72ºC. This is the enzyme of choice for daily or demanding RNA reverse transcription. OneScript® Hot is formulated with abm’s RNaseOFF Ribonuclease Inhibitor offering improved resistance to oxidation compared to the high oxidation-sensitive human RNase inhibitors. RNaseOFF is stable even under very low concentrations of DTT (< 1 mM), making it the best choice for ultimate RNA protection. Product Features:
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| Storage Condition |
Store at -20ºC. |
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| Material Citation | If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. G593 |
| Can I use both total RNA and poly(A)+ mRNA for cDNA synthesis? | |
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Yes, both total RNA and poly(A)+ mRNA can be used, though poly(A)+ mRNA typically yields higher quantities and better purity.
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| Can I synthesize cDNA from long RNA transcripts? | |
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Yes, for longer RNA transcripts, extend the 60°C incubation time to up to 30 minutes.
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| What can I use the synthesized cDNA for? | |
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The high-quality cDNA can be used in a variety of downstream applications, including gene expression analysis, cloning, and PCR-based assays. BlasTaq™ 2X qPCR Master Mix (Cat. No. G891) is well suited to downstream qPCR applications.
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| How should I store the synthesized cDNA? | |
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Store the synthesized first-strand cDNA at -20°C for long-term use.
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| Why am I getting low cDNA yields? | |
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Low cDNA yields can be caused by poor RNA integrity, contamination, or insufficient RNA input. To improve yield:
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| How much RNA template should I use for cDNA synthesis? | |
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We recommend using 1 ng to 2 μg of RNA per reaction.
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| What volumes of cDNA should I use for downstream PCR? | |
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Typically, use 1 μl of cDNA in a 25 μl PCR reaction. You can add up to 20% of the PCR volume (e.g., 5 μl in a 25 μl PCR), depending on your target and primers.
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What if input RNA samples are expected to have high levels of genomic DNA contamination?
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If input RNA samples are expected to have high levels of genomic DNA contamination, we recommend using abm’s All-In-One 5X RT MasterMix with gDNA Removal (Cat. No. G592).
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| What is the advantage of OneScript® Hot Reverse Transcriptase compared to OneScript® Plus Reverse Transcriptase? | |
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OneScript® Hot Reverse Transcriptase offers a significant advantage over enzymes that operate at lower temperatures (42-55ºC) due to its exceptional thermostability, allowing it to function effectively at 60-72ºC. This higher temperature improves efficiency by preventing RNA secondary structures and enabling cDNA synthesis from degraded or low-abundance RNA. Additionally, it is more resistant to inhibitors and contaminants, making it ideal for complex or impure RNA samples.
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Do I need an RNase inhibitor when using this product?
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All of abm's Reverse Transcriptases include RNaseOFF Ribonuclease Inhibitor, which protects RNA from degradation and is resistant to oxidation, even under low DTT concentrations. Therefore, there’s no need to add an external RNase inhibitor, as RNaseOFF provides optimal RNA protection during reverse transcription.
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| What is the source of this enzyme? | |
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The enzyme is produced recombinantly in E. coli, which has been engineered to express the enzyme gene. While the original gene may come from another organism, all production and purification occur using E. coli under controlled conditions.
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| Can this product be used for next-generation sequencing (NGS) library preparation? | |
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This product can be used for NGS library preparation, but additional processing steps are recommended. NGS workflows require double-stranded DNA, and the presence of RNA:cDNA hybrids may interfere with downstream applications. To ensure compatibility, we suggest treating the reaction with RNase H to remove any residual RNA, followed by PCR amplification to generate the necessary double-stranded DNA for NGS library construction. |
| Does OneScript® Hot Reverse Transcriptase have RNase H activity? | |
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No. The OneScript® Hot Reverse Transcriptase enzyme does not have RNase H activity, which means it does not degrade the RNA strand in RNA:cDNA hybrids. While this helps maintain cDNA yield and integrity during first-strand synthesis, it also means RNA:cDNA hybrids can persist unless an RNase H digestion step is performed.
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