SuperScript III 反转录酶 (SuperScript III Reverse Transcriptase)： Evolution of the SuperScript Reverse Transcriptases

With over thousands of references and citations, the SuperScript reverse transcriptase (RT) family is a widely used brand of enzyme for cDNA synthesis. This brand of reverse transcriptase is designed to reliably deliver high yields of full-length cDNA. To meet a growing need for sensitivity, SuperScript III RT has been engineered for a higher level of thermostability and a longer shelf life at 50°C.

图1. Performance-based evolution of SuperScript III reverse transcriptase. SuperScript III RT was developed to provide maximum sensitivity for real-time RT-PCR, microarray experiments, and other gene expression applications.

In addition to polymerase activity, reverse transcriptase possesses RNase H activity that degrades RNA in RNA/DNA hybrids; unfortunately, this is detrimental to the synthesis of cDNA from mRNA in vitro. In addition, RNase H activity diminishes the yield and size of cDNA by hydrolyzing the RNA template as polymerization proceeds.

Eliminating RNase H activity results in increased yields of full-length cDNA. SuperScript III RT has mutations that knock out the RNase H activity. The ability of SuperScript III to amplify cDNA from 353 bp to 12.3 kb is demonstrated in Figure 2.

图2. SuperScript III RT increases yields of full-length cDNA. SuperScript III RT was used to amplify 353 bp to 12.3 kb cDNA. The larger amplicons require 1 µg of RNA, likely due to the low abundance of full-length RNA and the difficulty in reading through a long transcript.

RNA transcripts exhibit significant secondary structure that must be denatured for efficient reverse transcription. At higher temperatures, RNA is less structured, but common reverse transcriptases (e.g., MuLV and AMV) are also inactive at temperatures greater than 45°C. SuperScript III RT has been engineered for high thermostability and has a half-life of 220 minutes at 50°C. This increases its ability to process RNA with secondary structures (Figure 3)

Figure 3. (above) Thermostability of SuperScript III RT. SuperScript III RT is significantly more thermostable than MMLV or SuperScript II RT. For example, at 55°C, MMLV and SuperScript II RT are both inactive after 5 min, while SuperScript III RT is still 80% active after 10 min.

Figure 4. (below) shows that both MMLV and SuperScript II RT have difficulty generating cDNA as temperature increases. However, SuperScript III RT is active at 55°C and is able to generate the highest yields of full-length cDNA, e.g., a 9.5 kb fragment.

The ideal reverse transcriptase will reverse transcribe even the least abundant mRNA within a pool, allowing single-cell transcript detection. The high sensitivity of SuperScript III RT is demonstrated with targets of varying sizes (Figure 5).

图5. The high sensitivity of SuperScript III RT.(A) SuperScript III RT can easily achieve a signal with 0.1 pg input of transcripts, e.g.,β-actin 354 and glyceraldehye-3-phosphate dehydrogenase (GAPDH). (B) In addition, SuperScript III RT is also capable of creating cDNA for PCR amplification from a single HeLa cell.

The data shown above support the use of SuperScript III reverse transcriptase as a high-quality reverse transcriptase for gene expression analysis, but how does it fare against the competition? We ran a head-to-head comparison with a popular reverse transcriptase and found that SuperScript III RT delivers increased cDNA yields, has higher sensitivity, and generates more full-length transcripts than the competitior’s enzyme (Figure 6). Independent data1 have also shown SuperScript III RT to give a higher yield of full-length cDNA compared to several commercially available reverse transcriptases.

图6. Compared to a competitor’s reverse transcriptase, SuperScript III Reverse Transcriptase produces more full-length cDNA and more representative cDNA from endpoint RT-PCR.