EpiScript™ Reverse Transcripta

Applications

• First-strand cDNA synthesis for subsequent PCR or real-time PCR.

EpiScript™ Reverse Transcriptase (EpiScript RT), an alternative to SuperScript® II Reverse Transcriptase, is a recombinant MMLV reverse transcriptase with reduced RNase H activity. It is highly efficient at producing full-length cDNA from long RNA templates. EpiScript RT demonstrates significantly more activity than other MMLV Reverse transcriptase enzymes. It is capable of producing cDNA from as little as 50 pg of total RNA for real-time RT-PCR (qRT-PCR) analysis and other applications.

The enzyme is available in 25,000 and 50,000 unit sizes at a concentration of 200 U/µl. The enzyme is supplied with a 10X Reaction buffer and 100 mM DTT.

Storage: Store only at -20°C in a freezer without a defrost cycle.

Storage Buffer: EpiScript RT is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 1 mM DTT, 0.1 mM EDTA, and 0.1% Triton® X-100.

Unit Definition: One unit of EpiScript RT catalyzes the incorporation of 1 nmol of dTTP into acid-insoluble material in 10 minutes at 37°C using saturating amounts of oligo(dT)-primed poly(rA) as template.

Contaminating Activity Assays: EpiScript RT is free of detectable exonuclease, endonuclease, and RNase activities.

Figure 1. EpiScript™ Reverse Transcriptase performed equally or better than comparable reverse transcriptases from other vendors in 2nd strand-qPCR reactions. First- strand synthesis reactions were assembled according to manufacturer´s specifications. Input RNA was 1 µg of Jurkat total RNA (Ambion®). Reactions were primed using 50 ng of poly-T(16-18) DNA. 2nd strand qPCR was performed using Bio-Rad iQ SYBR mastermix and gene-specific primers that yielded 250-350 bp amplicons. Reactions were repeated 4-fold. PGDF-R (Platelet Derived Growth Factor Receptor), TNF (Tumor Necrosis Factor), IL-1b (Interleukin-1 beta), IL-2 (Interleukin 2). Image courtesy of Matthew Kellinger, Illumina® Inc.

Figure 2 (click to enlarge). EpiScript™ Reverse Transcriptase produces similiar transcript coverage independent of transcript length. EpiScript was used to prime first strand cDNA either from total RNA using oligo-dT (top panel) or polyA+ selected RNA with random hexamers (bottom panel). The cDNA was converted into Illumina®-compatible libraries and sequenced. The reads were aligned to transcripts based upon various length classes and read density plotted relative to the percent distance from the 5´ end of the transcripts; 0% refers to the 5´ end and 100 % is the 3´ end. As expected, oligo-dT priming results in a more pronounced 3´ bias than random priming.

Figure 3. Use of EpiScript® Reverse Transcriptase for first strand cDNA results in detection of similar transcript categories independent of input amount. EpiScript RT was used to random prime 5 ng or 50 pg of human total RNA and the cDNA was converted into libraries for Illumina® sequencing. Reads were aligned using Tophat and annotated with Cufflinks and the percentage of each major category is visualized as a pie chart. Equivalent results are observed for both 5 ng and 50 pg of input RNA.