Reverse Transcription

SunScript™ Reverse Transcriptase

highly thermostable and efficient

SunScript™ is a novel, highly thermostable reverse transcriptase engineered from the well characterized HIV-1 RT. SunScript allows reaction temperatures up to 85ºC, offers improved efficiency of cDNA synthesis and comes in 2 different versions: +RNase H and –RNase H.

RNase H+: Engineered for high thermostability the enzyme is active up to temperatures of 85°C. It has RNA and DNA-dependent polymerase activities as well as RNase H activity.

RNase H-: Engineered for high thermostability and absence of RNase H activity the enzyme allows high yield, full length cDNA synthesis on long RNA molecules with complex secondary structures.

Note: SunScript™ RNaseH- is a key component in the SunScript™ One Step RT-PCR Kit.

Advantages

Highly thermostable: active up to 85ºC to resolve even the most complex RNA structures
High reaction speed of >30 bases per second
High yields of first strand cDNA
Complete reverse transcription of very long RNA molecules
High sensitivity (down to pgs of starting total RNA)
Yields highly consistent and reproducible results

Reaction temperatures up to 85ºC

100 ng of total RNA from HeLa cells were reverse transcribed at the indicated temperatures for 60 min with SunScript™ Reverse Transcriptases and Oligo(dT)18 primer, followed by inactivation at 95°C for 10 min. Presence of GAPDH cDNA was detected by PCR (product length 305 bp). High reaction temperatures are advantageous to transcribe RNAs with complicated secondary structures.

High Sensitivity Over Wide Range Of RNA Concentrations

Sensitivity test: RT reactions were performed at 70°C for 60 min. Inactivation at 95°C for 10 min. Primer set : Human GAPDH set (305bp). Sample: total RNA from HeLa cells. Concentrations: 1) 1 μg, 2) 100 ng, 3) 10 ng, 4) 1 ng, 5) 100 pg.

Detection Of Long Reads

Assay for long mRNA reverse transcription: 3 independent RT reactions were performed at 65°C for 60 min using Oligo(dT)20 primers. Inactivation at 95°C for 10 min. PCR Primer sets : mmNEB (>16kb from 3’end of mRNA; mRNA total length 22.5kb); mmDnah8 (>8,5kb from 3’end of mRNA; mRNA total length 14.5kb). Lanes 1-3 SunScript RT RNaseH+; Lanes 4-6 SunScript RT RNaseH-