StellarScript HT Highlights
- Increased thermostability enhances cDNA generation from RNA with a high degree of secondary structure
- Resistance to common inhibitors provides compatibility with a variety of sample sources and applications
- Improved activity enables reverse transcription from input amounts as low as 10 pg
- 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)
- 3' Rapid amplification of cDNA ends (RACE)
- Applications leveraging SuperScript III 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
Overcome secondary structure with a thermostable RT
It can be difficult to generate full-length cDNA from RNAs with a high degree of secondary structure, such GC-rich and viral RNAs. Reverse transcribing at elevated temperatures can relax this secondary structure, as well as reduce nonspecific primer hybridization.
StellarScript® HT generates higher cDNA yields and performs equivalently with respect to processivity relative to SuperScript III at elevated temperatures.
For maximum thermostability, see StellarScript HT+.
Overcome secondary structure with a thermostable RT
Increased resistance to common inhibitors
Inhibitors reduce cDNA synthesis efficiency and can adversely impact assay sensitivity. Improved inhibitor tolerance safeguards performance and provides compatibility across a wide range of sample types and sources.
StellarScript HT delivers superior inhibitor tolerance in comparison to StellarScript. For maximum inhibitor tolerance, see StellarScript HT+.
Figure 2. Reliable performance in the presence ... MORE
Increased resistance to common inhibitors
Figure 2. Reliable performance in the presence of common inhibitors. StellarScript, StellarScript HT, and StellarScript HT+ were run in oligo-dT-primed first strand synthesis for 30 minutes using 10 ng total liver RNA as template in varying concentrations of common inhibitors. Resulting cDNA yields were assessed via qPCR. StellarScript HT provides enhanced inhibitor tolerance in comparison to StellarScript.
Generate cDNA from vanishingly low RNA inputs
StellarScript HT robustly synthesizes cDNA from a broad range of RNA inputs, including as little as 10 pg. This enhances compatibility with applications where RNA template is limiting.
Figure 3. StellarScript HT provides equivalent ... MORE
Generate cDNA from vanishingly low RNA inputs
Figure 3. StellarScript HT provides equivalent performance to SuperScript III. Watchmaker's StellarScript HT and ThermoFisher Scientific's SuperScript III were run in oligo-dT and randomly primed first strand synthesis at 50°C for 40 minutes using 1000 ng to 0.01 ng total liver RNA as template. Resulting cDNA yields were assessed via qPCR. StellarScript HT showed overall equivalency to SuperScript III.
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 HT Reverse Transcriptase Kit User Guide
Enzymes MDx StellarScript HT RTStellarScript® HT Reverse Transcriptase enables efficient, high-specificity cDNA synthesis at 50°C, ideal for degraded RNA inputs and applications like RT-qPCR and RNA sequencing.
| CONFIGURATION | |
|---|---|
| Kit contents | StellarScript® HT 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 | |
|---|---|
| Purity (SDS-PAGE) | >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 HT Reverse Transcriptase Kit (200 U/µL) incl. 10X RT Reaction Buffer, 10 mM dNTP Mix, 100 mM DTT | 7K0070-50UL | 7K0070-200UL | 7K0070-1ML | Request a quote |
Please contact sales@watchmakergenomics.com to inquire about custom kit configurations.
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