SuperScript® III 反转录酶 (SuperScript® III Reverse Transcriptase)： Evolution of the SuperScript® Reverse Transcriptases
SuperScript® III逆转录酶 (RT) 可提供较其他逆转录酶更高的全长cDNA产量，具有更完整的基因代表性。 稳定的cDNA合成是基因表达研究的关键。 不恰当的逆转录 (RT) 反应会降低cDNA产量，从而影响反应的灵敏性，或者会形成缩短的cDNA，产生假阴性结果。 由于大多数逆转录酶的RNA酶H活性很高且热稳定性较低，因此无法利用起始RNA稳定地生成具有完整基因代表性的cDNA。 SuperScript® III逆转录酶在50°C下具有更高的热稳定性和更长的半衰期。 此外，SuperScript® III的RNA酶H活性较低。 如果您要获得最高的实时荧光定量RT-PCR和芯片实验的灵敏度，以及代表起始RNA的最高保真度的cDNA，那么您需要SuperScript® III逆转录酶。
进一步了解 SuperScript® III RT
Engineered for Performance
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.
Importance of RNase H
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.
Effects of Temperature on cDNA Synthesis
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)
Half-life (min) at 50o C
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.
RNA Detection Sensitivity
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.
SuperScript® III vs. the Competition
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.
cDNA Synthesis direct from cells
End-point cDNA Synthesis