StellarScript Highlights
- Increased activity generates cDNA from exceedingly low RNA inputs
- Template switching activity delivers compatibility with applications such as single-cell RNA-seq and 5' RACE
- Enhanced thermostability in comparison with wild-type enzyme improves reverse transcription through RNA secondary structure
- Kits contain all required reagents for cDNA synthesis, except primers, for simple experimental setup
- Custom formats available, including those to support lyophilization applications
Applications
- First strand cDNA synthesis
- Reverse transcription quantitative PCR (RT-qPCR)
- Reverse transcription PCR (RT-PCR)
- Primer extension
- RNA sequencing (RNA-seq)
- Single-cell RNA sequencing (scRNA-seq)
- 3' and 5' Rapid amplification of cDNA ends (RACE)
- Applications leveraging SuperScript II for reverse transcription
Want more information? Visit our other StellarScript portfolio product pages:
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| Property | Application | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Optimal Temp | Thermal Stability | Inhibitor Tolerance | Template Switching | RT-qPCR/ qRT-PCR | RT-PCR | RNA-seq | scRNA-seq | 5' RACE | 3' RACE | |
| StellarScript HT+ | 42 – 65˚C | +++ | +++ | + | +++ | +++ | + | + | + | + |
| StellarScript HT | 42 – 50˚C | ++ | ++ | – | ++ | ++ | + | – | – | + |
| StellarScript | 42˚C | + | + | + | + | + | + | + | + | + |
Table 1. Overview of StellarScript portfolio enzyme properties and relevant applications. "+" signs indicate strength of a property or fit of an application for a specific enzyme.
Key Performance Data
Achieve robust performance with low-input amounts
Many applications are limited in the available amount of input RNA, which can impact assay performance. StellarScript® generates high-quality cDNA across a wide range of RNA input amounts, delivering sensitive performance with very little RNA template.
Figure 1. StellarScript provides equivalent ... MORE
Achieve robust performance with low-input amounts
Figure 1. StellarScript provides equivalent performance to SuperScript II. Watchmaker's StellarScript and ThermoFisher Scientific's SuperScript II were run in oligo-dT and randomly primed first strand synthesis at 42°C for 40 minutes using 1000 ng to 0.01 ng total liver RNA as template. Resulting cDNA yields were assessed via qPCR. StellarScript showed equivalency with inputs of 1 ng or more, and showed slight outperformance with lower input amounts.
Improve specificity with a thermostable RT
StellarScript provides enhanced thermostability in comparison to wild-type M-MLV RT and performs equivalent-to-better in comparison to other commercially available M-MLV RT variants with respect to both cDNA yield and lengths generated at elevated temperatures. This improves performance with RNAs that have high degrees of secondary structure, including viral RNA and those that are GC-rich.
For further enhanced thermostability, see StellarScript HT and StellarScript HT+.
Improve specificity with a thermostable RT
BROCHURES
Precision Enzymes and Proteins Brochure
Custom Genomic Solutions Enzymes MDx DNA Polymerase I Equinox Library Amplification Equinox Uracil-Tolerant Library Amplification phi29 DNAP RNase Inhibitor StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA Polymerase T4 DNA Ligase T4 DNAP T4 Polynucleotide Kinase T4 Gene 32 Protein Taq DNAPWatchmaker Genomics offers a portfolio of precision-engineered enzymes and proteins, designed to meet the demands of high-stringency applications. We provide tailored services such as custom fills, formats, packaging, and labeling to accommodate unique specifications.
StellarScript® Reverse Transcriptase Portfolio Brochure
RT-PCR/RT-qPCR RNA Blood FFPE Microbial Saliva/buccal Enzymes MDx StellarScript RT StellarScript HT RT StellarScript HT+ RTStellarScript® Reverse Transcriptase is an engineered M-MLV variant with reduced RNase H activity, offering enhanced processivity, thermostability, and inhibitor tolerance for applications such as first-strand cDNA synthesis, RT-qPCR, and RNA sequencing.
POSTERS
Parallelized reverse transcription engineering and rapid whole transcriptome sequencing workflow optimization delivers improved gene detection sensitivity
Illumina RNA Blood Frozen Tissue Enzymes StellarScript RT StellarScript HT RT StellarScript HT+ RTThis poster details the development of engineered reverse transcriptases (RTs) and an optimized whole transcriptome sequencing workflow to enhance gene detection sensitivity, particularly with low-input and challenging samples like formalin-fixed, paraffin-embedded (FFPE) tissues.
Engineered reverse transcriptases and Taq DNA polymerases deliver robust yield in the presence of clinically relevant inhibitors
PCR/qPCR RT-PCR/RT-qPCR DNA RNA Enzymes MDx StellarScript RT StellarScript HT RT StellarScript HT+ RT StellarTaq DNA PolymeraseThis poster discusses the engineering of reverse transcriptases and Taq DNA polymerases to enhance thermostability and inhibitor tolerance, addressing challenges in molecular diagnostics. The engineered enzymes, such as StellarScript HT+ and aCat174 Taq DNA polymerase, demonstrate improved performance in the presence of common inhibitors, making them suitable for applications like RT-qPCR, RNA sequencing, and PCR amplification from challenging samples.
Purpose built reverse transcriptases and Taq DNA Polymerases deliver high yield in the presence of inhibitors
PCR/qPCR RT-PCR/RT-qPCR DNA RNA Cell Line Frozen Tissue Enzymes MDx StellarScript RT StellarScript HT RT StellarScript HT+ RTThis poster discusses the engineering of reverse transcriptases (RTs) and Taq DNA polymerases (Taq DNAPs) to enhance thermostability, inhibitor tolerance, and speed. The study identifies RT variants with increased thermostability and inhibitor tolerance, and Taq DNAP variants with enhanced speed and inhibitor tolerance, making them suitable for point-of-care applications.
PROTOCOLS
StellarScript Reverse Transcriptase Kit User Guide
Enzymes MDx StellarScript RTProtocol for StellarScript® Reverse Transcriptase enables efficient, specific cDNA synthesis at 42°C, supporting applications like RT-qPCR and RNA sequencing with intact or degraded RNA inputs.
| CONFIGURATION | |
|---|---|
| Kit contents | StellarScript® RT (200 U/µL), 10X RT Reaction Buffer, 10 mM dNTP Mix, 100 mM DTT |
| Unit definition | One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using poly(A)/oligo (dT) as a substrate |
| Enzyme storage buffer formulation | 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1% Tween 20, 50% glycerol |
| 10X RT Reaction Buffer formulation | 500 mM Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl₂ |
| QUALITY CONTROL | |
|---|---|
| Protein Purity Assay | >99% |
| dsDNA exonuclease | <1% released |
| ssDNA exonuclease | <1% released |
| DNA contamination (E. coli, mammalian, library) | <10 copies |
| Phosphatase assay | <1% released |
| Endonuclease | Not detectable |
| Nonspecific RNase | Not detectable |
| SHIPPING AND HANDLING | |
|---|---|
| Shipping conditions | Ice packs |
| Storage | -20°C ± 5°C |
| Shelf life | 12 months |
What is the StellarScript portfolio?
The StellarScript® portfolio includes three reverse transcriptase enzymes: StellarScript, StellarScript HT, and StellarScript HT+. StellarScript Reverse Transcriptases (RT) are M-MLV RT variants with improved thermostability and reduced RNase H activity. These enzymes have increased specificity, higher cDNA yields, and longer cDNA products than wild type M-MLV RT. StellarScript HT and HT+ have been engineered for increased thermostability and inhibitor tolerance. StellarScript and StellarScript HT+ have template switching activity.
What are potential applications for StellarScripts?
- First strand cDNA synthesis
- First strand cDNA synthesis at elevated temperatures and in the presence of inhibitors
- One- and two-step RT-qPCR
- Primer extension
- Single-cell RNA sequencing (for StellarScripts with template switching ability)
When should I use StellarScript, StellarScript HT or StellarScriptHT+
- StellarScript is ideal for applications that require low-temperature, first strand synthesis including some on-bead applications.
- StellarScript HT can be used for applications that do not require template switching that may need to be performed at 50°C.
- StellarScript HT+ should be used when inputs may contain inhibitors or high salt concentrations, when temperatures above 50°C are required or when template switching is necessary.
Can RNase inhibitors be used with StellarScripts?
RNase Inhibitor is not included in this kit and is not required for first strand synthesis, but the inclusion of RNase Inhibitor often increases cDNA yield. If the RNA input is from a crude sample or a sample with potential RNase contamination, an RNase Inhibitor is highly recommended.
Is DTT recommended for use?
No, but a minimum final concentration of 1 mM DTT is recommended in first strand synthesis. Certain RNase Inhibitors require additional DTT for optimal activity. Refer to manufacturer's instructions for DTT requirements.
What input types are StellarScripts compatible with?
StellarScript HT+ is appropriate for use with both intact RNA and degraded RNA, such as FFPE-derived, with inputs ranging from 10 pg to 1 µg.
How is RNase H activity turned off in StellarScript Enzymes?
The catalytic domain for RNase H for all the StellarScript enzymes is fully knocked out, but the RNaseH domain remains intact.
What storage buffer is used for the three StellarScripts?
The storage buffer and reaction buffer composition is the same across all three enzymes, and is published in our user guides.
Can StellarScript template switch?
StellarScript and StellarScript HT+ have the best ability to template switch.
Which StellarScript has the best inhibitor tolerance?
StellarScript HT+ has the best inhibitor tolerance, with the least inhibition in PCR.
What are general considerations for RT performance?
The optimal temperature for StellarScript is 42°C. The optimal temperature for HT and HT+ is 50°C. If your application requires you to go above 50°C, then we recommend using HT+.
How can secondary structures be removed from the RNA?
Reaction temperatures of 42 – 50°C relaxes secondary structures of RNA targets, increasing the length of products.
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| Description | 10 kU | 40 kU | 200 kU | |
|---|---|---|---|---|
| StellarScript® Multi Reverse Transcriptase Sample Pack (200 U/µL) incl. 10X RT Reaction Buffer, 10 mM dNTP Mix, 100 mM DTT, and 50 µL each of StellarScript RT, StellarScript HT RT, and StellarScript HT+ RT | 7K0083-50UL | - | - | Request a quote |
| StellarScript Reverse Transcriptase Kit (200 U/µL) incl. 10X RT Reaction Buffer, 10 mM dNTP Mix, 100 mM DTT | 7K0080-50UL | 7K0080-200UL | 7K0080-1ML | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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