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.
If StellarScript HT+ is active from 42 – 65°C, at what temperature should I run my reaction?
To decrease RNA secondary structures, we recommend a first strand synthesis temperature from 50 – 55°C. Temperatures > 55°C should only be run when it is required for your application.