STELLARSCRIPT HT+ REVERSE TRANSCRIPTASE FAQs

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.

• 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)

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.

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.

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.

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.

The catalytic domain for RNase H for all the StellarScript enzymes is fully knocked out, but the RNaseH domain remains intact.

The storage buffer and reaction buffer composition is the same across all three enzymes, and is published in our user guides.

StellarScript and StellarScript HT+ have the best ability to template switch.

StellarScript HT+ has the best inhibitor tolerance, with the least inhibition in PCR.

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+.

Reaction temperatures of 42 – 50°C relaxes secondary structures of RNA targets, increasing the length of products.

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.